Head and Neck Cancer: Multimodality Management

Head and Neck Cancer

Jacques Bernier Editor

Head and Neck Cancer Multimodality Management

Editor Jacques Bernier, MD, PD Department of Radio-Oncology Genolier Swiss Medical Network Geneva, Switzerland and Faculty of Medicine University of Geneva Geneva, Switzerland [email protected]

ISBN 978-1-4419-9463-9 e-ISBN 978-1-4419-9464-6 DOI 10.1007/978-1-4419-9464-6 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011928238 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

For the last two decades, the management of the head and neck cancers has been characterized by a number of profound mutations, both triggered by the willingness of oncologists to reshape significantly their conceptual approaches to locally advanced diseases and facilitated by the advent of new, more active drugs. Indeed, the potentialities of functional imaging, the significant progresses in conservative surgery and reconstruction, the acknowledged role of concurrent chemo-radiation, the investigational use of combined therapies for organ preservation, and the advent of targeted therapies are among the main tracks along which fundamental changes in philosophy of management have been recently observed. Coincident with this came the necessity to move progressively to a much more holistic approach, taking greater account of quality of life after treatment. At the same time, it is important to remember that progress in head and neck cancer management is benefiting from an increasing understanding of all aspects involved in the development of these carcinomas, ranging from the molecular genetics of tumor growth to epidemiological and etiological factors, the role of which has been somewhat underestimated in the past, especially with respect to the natural history of the disease in individual patients. Recently, intensive translational research in head and neck oncology yielded a large burden of significant laboratory discoveries and clinical breakthroughs. Paradoxically, these advances generated at the same time an often contradictory and confusing body of literature, which nevertheless emphasizes that, as red thread, the management of this disease must be multidisciplinary. Therefore, there is room for a guide to contemporary management of head and neck cancer, and with this perspective, this textbook was designed and written with the intent of providing oncologists with a broad, comprehensive and balanced view of both consolidated and innovative concepts. Its objective is to help practitioners understand better the mechanisms that drive the development of head and neck cancer, as well as revisit the evidence-based data and complexities of decision-making in this field. The first part of the textbook covers the vast domain that pertains to the scientific basis of head and neck oncology, ranging from epidemiology and etiology of these carcinomas to biomolecular mechanisms driving the cell malignant transformation and tumor growth. In particular, this book provides key information on those molecular analyses that are bound to pave the way, in head and neck oncology, for the eventual development of highly personalized future treatment strategies. The second part reviews the key diagnosis modalities used in head and neck oncology, a field in which great progress is being made with not only a refinement of morphological imaging, but also an intensive preclinical and clinical research devoted to tumor metabolism. A greater understanding of the correlation between tumor types and patterns of spread will indeed allows clinicians to optimize combinations of treatments in each individual patient.

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Preface

The book continues with a systematic account of the current management of individual cancers in function of their site of origin. The chapters of this third section of the textbook are organized to provide the readers with an exhaustive review of organ-oriented strategies. Each contribution explores the most recent evidence-based data and expert opinions, which reflect up-to-date diagnosis and treatment approaches. The fourth and last part covers a number of issues worth considering in the comprehensive management of this disease, such as, quality assurance programs, patient rehabilitation, salvage treatments, and quality of life. This textbook is aimed at showing how considerable are today’s efforts toward improving treatment outcomes for head and neck cancer patients. This willingness to make significant and rapid progress probably derives from the fact that rarely in oncology are treatment failures more readily visible and functionally impairing than in these patients. This, together with the fact that head and neck cancer is a “big killer” worldwide, fully justifies the intensive translational and clinical research that fosters and optimizes our ways to plan and deliver treatments. Undoubtedly, these efforts allow investigators to take a number of significant steps forward, which foreshadow the implementation of treatment combinations tailored to each patient. A timely account of our present knowledge of the principles and present day practice, this textbook reflects the considerable personal experiences of the experts who offer their contributions. Without access to a multidisciplinary tumor board for guidance, many practitioners often remain confused about the appropriate therapy for patients with head and neck cancer. In this perspective too, we hope that this textbook will provide a source of knowledge and references not only to the established oncologist entrusted with the care of head and neck cancer patients, but also to the less experienced practitioner willing to get a better understanding of this multifaceted, complex field of oncology. Geneva, Switzerland

Jacques Bernier

Contents

  1 Epidemiology and Aetiology of Head and Neck Cancers..................................... Newell W. Johnson and Hemantha K. Amarasinghe

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  2 Head and Neck Cancer Prevention........................................................................ Fausto Chiesa, Angelo Ostuni, Roberto Grigolato, and Luca Calabrese

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  3 Cellular and Molecular Pathology of Head and Neck Tumors............................ Adel K. El-Naggar

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  4 Oncogenomics/Proteomics of Head and Neck Cancers........................................ Howard S. Moskowitz and Jennifer R. Grandis

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  5 Genetics and Epigenetics of Head and Neck Cancer............................................ Richard J. Shaw and Jagtar Dhanda

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  6 Immunology of Head and Neck Cancer................................................................. 107 Steve C. Lee and Robert L. Ferris   7 HPV and EBV in Head and Neck Cancer............................................................. 121 Jeffrey Brumbaugh, Robert L. Ferris, and Shen Hu   8 Head and Neck Cancer Staging and Prognosis: Perspectives of the UICC and the AJCC............................................................... 135 Brian O’Sullivan and Jatin P. Shah   9 Biomarkers in Head and Neck Cancer.................................................................. 157 John Wrangle, Shanthi Marur, and Arlene A. Forastiere 10 Hypoxia in Head and Neck Cancers: Clinical Relevance and Treatment.......... 169 Yungan Tao and Jean Bourhis 11 Translational Research in Head and Neck Oncology........................................... 179 David S. Yoo and David M. Brizel 12 Preclinical Models of Head and Neck Squamous Cell Carcinoma...................... 191 Michiel W.M. van den Brekel, C.L. Zuur, Stephen P. Malkoski, and Xiao-Jing Wang 13 Morphologic Investigations in Head and Neck Cancer........................................ 201 Suyash Mohan and Gaurang V. Shah

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14 Ultrasound Investigations in Head and Neck Cancer Patients............................ 221 Yolanda Y.P. Lee, K.T. Wong, Ann D. King, and Anil T. Ahuja 15 Functional Imaging.................................................................................................. 235 Ludwig G. Strauss and Antonia Dimitrakopoulou-Strauss 16 Sentinel Node Biopsy for Head and Neck Cancer................................................. 241 Lee Alkureishi and Gary L. Ross 17 Approaches to Supportive Care.............................................................................. 255 Barbara A. Murphy 18 Intensity-Modulated Radiation Therapy for Head and Neck Cancer................ 267 Nancy Lee, Daniel Gomez, Edward J. Shin, and K.S. Clifford Chao 19 Principles of Systemic Chemotherapy for Squamous Cell Head and Neck Cancer...................................................................................................... 281 Cristina P. Rodriguez and David J. Adelstein 20 Molecular Targeted Therapies in Head and Neck Cancer................................... 293 Vinai Gondi, Anne M. Traynor, and Paul M. Harari 21 Phase I Study Methodology in Head and Neck Oncology.................................... 307 Christophe Le Tourneau and Lillian L. Siu 22 Evidence-Based Head and Neck Oncology: Principles and Pitfalls.................... 317 Søren M. Bentzen 23 Programs of Organ and Function Preservation.................................................... 327 Jean-Louis Lefebvre 24 Multidisciplinary Approach of Unresectable Head and Neck Cancer................ 333 Ricardo Hitt, Blanca Homet, and Robert I. Haddad 25 Laser Endoscopic Treatment.................................................................................. 339 Pierre R. Moreau and Pierre H. Demez 26 Multidisciplinary Management of Skull Base and Superstructure Tumors...... 351 Giulio Cantù, Carlo L. Solero, Stefano Riccio, Sarah Colombo, and Madia Pompilio 27 Multidisciplinary Management of Oral Cavity and Maxillary Sinus Cancers........................................................................................................... 363 Alexander D. Rapidis 28 Management of Nasopharyngeal Carcinoma........................................................ 381 Anne W.M. Lee, Wai-Tong Ng, Tsz-Kok Yau, Stephen C.K. Law, and William I. Wei 29 Multidisciplinary Management of Oropharynx Carcinomas.............................. 401 Beth M. Beadle and David I. Rosenthal 30 Multidisciplinary Management of Hypopharyngeal Carcinoma........................ 431 Marc Hamoir, Jean-Pascal Machiels, Sandra Schmitz, and Vincent Gregoire

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Contents

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31 Cancers of the Larynx: Tis, T1, T2 Evaluation and Management...................... 457 Carol M. Lewis, Ollivier Laccourreye, Randal S. Weber, and F. Christopher Holsinger 32 Diagnosis and Multidisciplinary Treatment of Laryngeal Cancers.................... 471 Nabil F. Saba, J. Trad Wadsworth, Jonathan J. Beitler, and Fadlo R. Khuri 33 Principles and New Approaches in Surgical Reconstruction............................... 483 Christina Kenney Magill and Bruce H. Haughey 34 Multidisciplinary Treatment of the Neck............................................................... 497 Remco de Bree, Johannes A. Langendijk, and C. René Leemans 35 Postoperative Management of High-Risk Resectable Head and Neck Cancer...................................................................................................... 513 Jay S. Cooper 36 Management of Salivary Gland Cancer................................................................ 521 Laura D. Locati, Marco Guzzo, Patrizia Olmi, and Lisa Licitra 37 Head and Neck Melanoma...................................................................................... 533 Genevieve A. Andrews and Jeffery N. Myers 38 Cervical Lymph Node Metastases of Squamous Cell Carcinoma from an Unknown Primary Site............................................................................. 547 Nicholas Pavlidis and George Plataniotis 39 Management of Thyroid and Parathyroid Cancers.............................................. 555 Ranee Mehra, Charu Aggarwal, and Roger B. Cohen 40 Head and Neck Paragangliomas............................................................................. 569 Matthew O. Old and James L. Netterville 41 Treatment of the Elderly Head and Neck Cancer Patient................................... 581 Jean-Claude Horiot and Matti S. Aapro 42 Interstitial Radiation Therapy in Cancer of the Oropharynx and Oral Cavity........................................................................................................ 589 Peter C. Levendag, David N. Teguh, and Paul I.M. Schmitz 43 Proton Beam Therapy for Head and Neck Cancer............................................... 605 Danielle N. Margalit, Judy A. Adams, and Annie W. Chan 44 Normal Tissue Complications and Protection in Head and Neck Cancer Patients....................................................................................... 613 Andy Trotti, Nikhil Rao, Avraham Eisbruch, and David I. Rosenthal 45 Rehabilitation of Heavily Treated Head and Neck Cancer Patients................... 629 Rachael E. Kammer and JoAnne Robbins 46 Salvage Therapy in Head and Neck Cancer Patients........................................... 641 Saba Aftab, Rod Rezaee, and Pierre Lavertu

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47 Systemic Treatment of Recurrent/Metastatic Squamous Cell Carcinoma of the Head and Neck........................................................................... 651 Jan B. Vermorken 48 Best Supportive Care for Palliative Treatment..................................................... 665 Michaela Salzwimmer 49 Quality of Life in Head and Neck Cancer Patients............................................... 675 Jolie Ringash 50 Quality Assurance of Clinical Trials in the Management of Cancer in the Head and Neck............................................................................................... 687 Thomas J. FitzGerald, Maryann Bishop-Jodoin, Marcia M. Urie, Kenneth Ulin, Geoffrey S. Ibbott, James A. Purdy, Joel H. Saltz, Lester J. Peters, M. Giulia Cicchetti, Mitchell Schnall, Joseph Deasy, Walter R. Bosch, Michael V. Knopp, Lawrence H. Schwartz, Keith S. White, Richard Hanusik, Sandra K. Kessel, Karen Morano, and Fran Laurie Index.................................................................................................................................. 695

Contents

Contributors

Matti S. Aapro, MD IMO (Multidisciplinary Oncology Institute), Clinique de Genolier, Vaud, Switzerland Judy A. Adams, CMD Department of Radiation Oncology, Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA David J. Adelstein, MD Department of Solid Tumor Oncology, Cleveland Clinic Taussig Cancer Center, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA Saba Aftab, MD Department of Otolaryngology – Head and Neck Surgery, University Hospitals Case Medical Center, Cleveland, OH, USA Charu Aggarwal, MD Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA Anil T. Ahuja, MD, FRCR Department of Diagnostic Radiology and Organ Imaging, The Chinese University of Hong Kong, Hong Kong, China Lee Alkureishi, MRCS, MBChB Department of Plastic and Reconstructive Surgery, University of Chicago, Chicago, USA Hemantha K. Amarasinghe, BDS, MSc, MD School of Dentistry and Oral Health, University of Griffith, Southport, QLD, Australia Genevieve A. Andrews, MD Department of Head and Neck Surgery, UT MD Anderson Cancer Center, Houston, TX, USA Beth M. Beadle, MD, PhD Radiation Oncology Department, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA Jonathan J. Beitler, MD, MBA, FACR Department of Radiation Oncology and Otolaryngology, Emory University School of Medicine, Atlanta, GA, USA Søren M. Bentzen, PhD, DSc Department of Human Oncology, Medical Physics, and Biostatistics and Medical Informatics, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA

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Jacques Bernier, MD, PD Department of Radio-Oncology, Genolier Swiss Medical Network, Geneva, Switzerland; University of Geneva, Geneva, Switzerland Maryann Bishop-Jodoin, BS Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Walter R. Bosch, DSc Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Jean Bourhis, MD, PhD Department of Radiotherapy, Institut Gustave Roussy, Villejuif, France Remco de Bree, MD, PhD Otolaryngology – Head and Neck Surgery Department, VU University Medical Center, Amsterdam, The Netherlands Michiel W.M.van den Brekel, MD, PhD Otorhinolaryngology – Head and Neck Surgery Department, Netherlands Cancer Institute – Antoni van Leeuwenhock Hospital and Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands David M. Brizel, MD Department of Radiation Oncology, Duke University Medical Center, Morris Building, Science Drive, Durham, NC, USA Jeffrey Brumbaugh School of Dentistry, UCLA, Los Angeles, CA, USA Luca Calabrese, MD Department of Head and Neck Surgery, European Institute of Oncology, Milano, Italy Giulio Cantù, MD Department of Cranio-Maxillo-Facial Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, National Cancer Institute, Via Venezian 1, Milano, Italy Annie W. Chan, MD Department of Radiation Oncology, Harvard Medical School, Boston, MA, USA K.S. Clifford Chao, MD Department of Radiation Oncology, Columbia University College of Physicians and Surgeons, New York, NY, USA; Division of Radiation Oncology, Weill Cornell College of Medicine, New York Presbyterian Hospital, New York, NY, USA Fausto Chiesa, MD Department of Head and Neck Surgery, European Institute of Oncology, Milano, Italy M. Giulia Cicchetti, MD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Roger B. Cohen, MD Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA Sarah Colombo, MD Department of Cranio-Maxillo-Facial Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, National Cancer Institute, Milano, Italy

Contributors

Contributors

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Jay S. Cooper, MD, FACR, FACRO, FASTRO Maimonides Cancer Center, Brooklyn, NY, USA Joseph Deasy, PhD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Pierre H. Demez, MD Department of Otorhinolaryngology, University Hospital of Liege, Liege, Belgium Jagtar Dhanda, BSc(Hons), MFDSRCS, MRCS Oral and Maxillofacial Surgery, University Hospital Birmingham, Birmingham, UK Antonia Dimitrakopoulou-Strauss, MD CCU Nuclear Medicine, German Cancer Research Center, Heidelberg, BW, Germany Avraham Eisbruch, MD Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA Adel K. El-Naggar, MD, PhD Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Robert L. Ferris, MD, PhD University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA Thomas J. FitzGerald, MD, BS Department of Radiation Oncology, University of Massachusetts Medical School, UMass Memorial Medical Center, QARC, Suite 201, Lincoln, RI, USA Arlene A. Forastiere, MD Oncology Department, Johns Hopkins University, School of Medicine, Bunting/Blaustein Cancer Research Building, Baltimore, MD, USA Daniel Gomez, MD Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Vinai Gondi, MD Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison, WI, USA Jennifer R. Grandis, MD Eye & Ear Institute, Pittsburgh, PA, USA Vincent Gregoire, MD, PhD, Hon. FRCR Radiation Oncology, Centre du Cancer, Universite Catholique de Louvain – Cliniques Universitaires Saint-Luc, Brussels, Belgium Roberto Grigolato Department of Head and Neck Surgery, European Institute of Oncology, Milano, Italy Marco Guzzo, MD Board Certified in Otorhinolaryngology and Maxillo Facial Surgery, Department of Surgery, Fondazione IRCCS-Istituto Nazionale Dei Tumori, Milan, Italy Robert I. Haddad, MD Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

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Marc Hamoir, MD Head and Neck Surgery, Universite Catholique de Louvain, St. Luc University Hospital and Cancer Center, Brussels, Belgium Richard Hanusik, AB Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Paul M. Harari, MD Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison, WI, USA Bruce H. Haughey, MBChB Department of Otolaryngology, Head and Neck Surgery, Washington University School of Medicine, St. Louis, MO, USA Ricardo Hitt, MD, PhD Medical Oncology, 12 de Octubre Madrid, Madrid, Spain F. Christopher Holsinger, MD Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Blanca Homet, MD Medical Oncology, 12 de Octubre Madrid, Madrid, Spain Jean-Claude Horiot, MD, PhD Clinique de Genolier, Institut Multidisciplinaire d’Oncologia, Genolier, Vaud, Switzerland Shen Hu, PhD School of Dentistry and Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA Geoffrey S. Ibbott, PhD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Newell W. Johnson, MDSc, PhD, FDSRCS, FRACDS, FCRPath, FFOP(RCPA), FMedSci Griffith Health Institute and School of Dentistry and Oral Health, Griffith University, Queensland, Australia Rachael E. Kammer, MS, CCC-SLP, BRS-S Department of Surgery, University of Wisconsin, Madison, WI, USA Sandra K. Kessel, BA Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Fadlo R. Khuri, MD Department of Hematology and Medical Oncology, Emory University, Atlanta, GA, USA Ann D. King, MRCP, FRCR Department of Diagnostic Radiology and Organ Imaging, The Chinese University of Hong Kong, Hong Kong, China Michael V. Knopp, MD, PhD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Ollivier Laccourreye, MD Serviced’Oto-Rhino-Laryngologie et Chirurgie Cervico-Faciale, Hôpital Européen Georges-Pompidou, Assistance Publique des Hôpitaux de Paris, Paris, France

Contributors

Contributors

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Johannes A. Langendijk, MD, PhD, MSc Department of Radiation Oncology, University Medical Center Groningen, Groningen, The Netherlands Fran Laurie, BS Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Pierre Lavertu, MD, FRCS©, FACS Department of Otolaryngology, Head and Neck Surgery, University Hospitals Case Medical Center, Cleveland, OH, USA Stephen C.K. Law, MBBS, DMRT, FRCR, FHKCR, FHKAM(Radiology) Clinical Oncology, Queen Elizabeth Hospital, Hong Kong, China Anne W.M. Lee, MD, FRCR Clinical Oncology, Pamela Youde Nethersole Eastern Hospital, Chai Wan, Hong Kong, China Nancy Lee, MD Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Steve C. Lee, MD, PhD Otolaryngology – Head and Neck Surgery, Loma Linda University, Loma Linda, CA, USA Yolanda Y.P. Lee, MBChB, FRCR Department of Diagnostic Radiology and Organ Imaging, The Chinese University of Hong Kong, Hong Kong, China C. René Leemans, MD, PhD Otolaryngology/Head and Neck Surgery, VU University Medical Center, Amsterdam, The Netherlands Jean-Louis Lefebvre, MD Head and Neck Department, Centre Oscar Lambret, Lille, France Peter C. Levendag, MD, PhD Department of Radiation Oncology, Erasmus MC – Daniel den Hoed Cancer Center, Rotterdam, The Netherlands Carol M. Lewis, MD, MPH Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Lisa Licitra, MD Department of Medical Oncology, IRCCS Fondazione Istituto Nazionale Tumori, Milan, Italy Laura D. Locati, MD Department of Medical Oncology, Fondazione IRCCS-Istituto Nazionale Dei Tumori, Milan, Italy Jean-Pascal Machiels, MD, PhD Medical Oncology, Centre du Cancer Universite Catholique de Louvain – Cliniques Universitaires Saint-Luc, Brussels, Belgium Christina Kenney Magill, MD Department of Otolaryngology, Head and Neck Surgery, Washington University School of Medicine, St. Louis, MO, USA Stephen P. Malkoski, MD, PhD Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Anschutz Medical Center, Aurora, CO, USA

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Danielle N. Margalit, MD, MPH Department of Radiation Oncology, Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA Shanthi Marur, MD, MBBS Oncology Department, Johns Hopkins University, School of Medicine, Baltimore, MD, USA Ranee Mehra, MD Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA Suyash Mohan, MD Radiology Department, University of Michigan, Ann Arbor, MI, USA Karen Morano, CMD Quality Assurance Review Center, University of Massachusetts Medical School, Lincoln, RI, USA Pierre R. Moreau, MD, PhD Department of Otorhinolaryngology, University Hospital of Liege, Liege, Belgium Howard S. Moskowitz, MD, PhD Otolaryngology Department, University of Pittsburgh Medical Center, Pittsburgh, PA, USA Barbara A. Murphy, MD Pain and Symptom Management Program, Internal Medicine, Vanderbilt Ingram Cancer Center, Nashville, TN, USA Jeffrey N. Myers, MD, PhD, FACS Department of Head and Neck Surgery, UT MD Anderson Cancer Center, Houston, TX, USA James L. Netterville, MD Otolaryngology and Hearing & Speech Sciences, Vanderbilt Bill Wilkerson Center, Nashville, TN, USA Wai-Tong Ng, MD, FRCR Clinical Oncology, Pamela Youde Nethersole Eastern Hospital, Chai Wan, Hong Kong, China Brian O’Sullivan, MD, FRCPC, FRCPI, FFRFRCSI (Hon) Head and Neck Oncology Program, Department of Radiation Oncology, Princess Margaret Hospital, Toronto, ON, Canada Matthew O. Old, MD Department of Otolaryngology – Head and Neck Surgery, The James Cancer Hospital and Solove Research Institute at the Ohio State University Comprehensive Cancer Center, Columbus, OH, USA Patrizia Olmi, MD Radiotherapy Unit, Imaging Diagnostic, Nuclear Medicine and Radiation Therapy Department, Fondazione IRCCS-Istituto Nazionale Dei Tumori, Milan, Italy Angelo Ostuni, MD Department of Head and Neck Surgery, European Institute of Oncology, Milano, Italy Nicholas Pavlidis University of Ioannina, Av. S. Niarchos, Ioannina, Greece Lester J. Peters, MD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA

Contributors

Contributors

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George Plataniotis, MD, PhD Oncology Department, Aberdeen Royal Infirmary, Aberdeen, UK Madia Pompilio, MD Department of Cranio-Maxillo-Facial Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, National Cancer Institute, Milano, Italy James A. Purdy, PhD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Nikhil Rao, MD Radiation Oncology Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Alexander D. Rapidis, MD, DDS, PhD, FACS Eastman Dental Institute, University College London, London, UK; Department of Head and Neck/Maxillofacial Surgery, Greek Anticancer Institute, Sant Savvas Hospital, Athens, Greece Rod Rezaee, MD, FACS Department of Otolaryngology – Head and Neck Surgery, University Hospitals Case Medical Center, Cleveland, OH, USA Stefano Riccio, MD Department of Cranio-Maxillo-Facial Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, National Cancer Institute, Milano, Italy Jolie Ringash, BSc, MD, MSc, FRCP© Department of Radiation Oncology, The Princess Margaret Hospital, Toronto, ON, Canada JoAnne Robbins, PhD Department of Medicine, University of Wisconsin, Madison, WI, USA; Geriatric Research Education and Clinical Center (GRECC), William S. Middleton Memorial Veterans Hospital, Madison, WI, USA Cristina P. Rodriguez, MD Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA David I. Rosenthal, MD Radiation Oncology Department, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA Gary L. Ross, MBChB, MD, FRCS(Plast) Plastic Surgery Department, The Christie, Manchester, UK Nabil F. Saba, MD, FACP Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA; Aerodigestive Malignancies Program, Winship Cancer Institute, Emory University, Atlanta, GA, USA Joel H. Saltz, MD, PhD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Michaela Salzwimmer, MD Department of General ORL, H&NS, Medical University of Graz, Graz, Austria Paul I.M. Schmitz, PhD Biostatistics, Erasmus MC – Daniel den Hoed Cancer Center, Rotterdam, The Netherlands

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Sandra Schmitz, MD Head and Neck Surgery, Universite Catholique de Louvain, St Luc University Hospital and Cancer Center, Brussels, Belgium Mitchell Schnall, MD, PhD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Lawrence H. Schwartz, MD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Gaurang V. Shah, MD Radiology Department, University of Michigan, Ann Arbor, MI, USA Jatin P. Shah, MD, PhD (Hon.), FACS, FRCS (Hon.), FRACS (Hon.), FDSRCS (Hon.) Head and Neck Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Richard J. Shaw, MD, FRCS, FDS Head and Neck Surgery, University of Liverpool, Liverpool, UK; Oral & Maxillofacial Surgery, University Hospital Aintree, Liverpool, UK Edward J. Shin, MD Otolaryngology, New York Eye and Ear Infirmary, New York, NY, USA Lillian L. Siu, MD Division of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, ON, Canada Carlo L. Solero, MD Neurosurgery, Fondazione IRCCS Istituto Neurologico “C. Besta”, Milan, Italy Ludwig G. Strauss, MD CCU Nuclear Medicine, German Cancer Research Center, Heidelberg, BW, Germany Yungan Tao, MD, PhD Department of Radiotherapy, Institut Gustave Roussy, Villejuif, France David N. Teguh, MD Department of Radiation Oncology, Erasmus MC – Daniel den Hoed Cancer Center, Rotterdam, The Netherlands Christophe Le Tourneau, MD Division of Medical Oncology, Institut Curie Paris, France Anne M. Traynor, MD Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison, WI, USA Andy Trotti, MD Radiation Oncology Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Kenneth Ulin, PhD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA Marcia M. Urie, PhD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA

Contributors

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Jan B. Vermorken, MD, PhD Medical Oncology Department, Antwerp University Hospital, Edegem, Belgium J. Trad Wadsworth, MD Otolaryngology – Head and Neck Surgery, Emory University, Atlanta, GA, USA Xiao-Jing Wang, MD, PhD Department of Pathology, University of Colorado, Denver, Aurora, CO, USA Randal S. Weber, MD, FACS Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA William I. Wei, MS, FRCS, FRCSE, FRACS(Hon), FACS, FHKAM(ORL)(SURG) Surgery, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China Keith S. White, MD Quality Assurance Review Center, Radiation Oncology, University of Massachusetts Medical School, Lincoln, RI, USA K.T. Wong, MBChB, FRCR Department of Diagnostic Radiology and Organ Imaging, The Chinese University of Hong Kong, Hong Kong, China John Wrangle, MD, MPH Oncology Department, Johns Hopkins University, School of Medicine, Baltimore, MD, USA Tsz-Kok Yau, MD, FRCR Clinical Oncology, Pamela Youde Nethersole Eastern Hospital, Chai Wan, Hong Kong, China David S. Yoo, MD, PhD Department of Radiation Oncology, Duke University Medical Center, Durham, NC, USA C.L. Zuur, MD, PhD Head and Neck Surgery, Oncology, The Dutch Cancer Institute – Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands

Chapter 1

Epidemiology and Aetiology of Head and Neck Cancers Newell W. Johnson and Hemantha K. Amarasinghe

Abstract  Malignant neoplasms of the head and neck are among the most common in the world and constitute a major public health problem in most countries. Over 90% of these are squamous cell carcinomas arising in the mucous membranes of the upper aerodigestive tract (UADT). Their epidemiology and aetiology are considered in detail. We separate nasopharyngeal cancer, because it has a specific aetiology related to Epstein-Barr Virus (EBV) infection and dietary carcinogens. We then add those sites with the common major risk factors of alcohol, tobacco (including betel quid/areca nut habits) and diets poor in antioxidants and vitamins, and a minor role for Human Papillomavirus (HPV). Collectively, these UADT sites of oral cavity (including tongue), other pharynx, and larynx have a male incidence/mortality of 15.2/8.1 and for females of 4.6/2.4 cases per 100,000 pa. This ranks UADT cancer as the sixth most common site for men, eighth for women. Adding nasopharynx pushes head and neck cancer higher up the scale. If oesophagus were to be included as another alcohol and tobacco-related cancer, the rates add to 28.6/18.9 and 10.1/6.8 respectively. These cancers – which might be termed cancers of the mouth, throat and gullet – then rank second only to lung cancer in men, and fourth after breast, uterine cervix and large bowel in females, worldwide. Detailed data are presented on geographical, ethnic, gender and time differences. The highest rates in the world are found in Melanesia, South Asia, parts of France, and much of Eastern Europe and the former Soviet republics. Many of these areas are showing rising trends, with a shift to involvement of younger individuals. This, and the fact that survival rates have improved little or not at all in much of the world over several decades, emphasises the need for effective primary and secondary prevention strategies – and for improved public policy to implement these.

N.W. Johnson (*) Griffith Health Institute and School of Dentistry and Oral Health, Building GO5, Room 3.22a, Gold Coast Campus, Griffith University, Queensland 4222, Australia e-mail: [email protected]

Keywords  Cancer • Head and neck • Upper aerodigestive tract • Mouth • Larynx • Nasopharynx • Tobacco • Alcohol • Nutrition • Human papillomavirus (HPV)

Introduction and Scope The term Head and Neck [H&N] Cancer is usually taken to cover the range of malignant neoplasms of soft tissue origin that develop in the oral cavity including the lips, nasal cavity, paranasal sinuses, pharynx, larynx and salivary glands. The skin will be included in many descriptions, but not usually ocular and intracranial neoplasms, nor those of endocrine or lymphatic origin – thus excluding thyroid and parathyroid cancers, and lymphomas. Sarcomas, though more rare, must be included among these soft tissue neoplasms of the head and neck, be they of connective tissue, neural or vascular origin. Summary data will be given on primary bone “tumours” and on those of odontogenic origin, though their pathology and management are not covered in detail in this volume. Readers are referred to the several excellent modern textbooks of surgical pathology and of oral and maxillofacial pathology: especially recommended are Fletcher DEM, Ed, Diagnostic Histopathology of Tumours, 3rd Edn., Elsevier 2007 and Gnepp DR, Ed., Diagnostic Surgical Pathology of the Head and Neck, 2nd Edn. Elsevier 2009. Reliable concise accounts created by a team of international experts appear in the series of WHO “blue books”, viz: Pathology and Genetics of Head and Neck Tumours, Brown L et  al. Eds., IARC Press, 2005. Metastases from distant primaries to the jaws (and occasionally to mucous membranes), must always be considered. Most head and neck cancers, indeed 95% or more, are squamous cell carcinomas (SCC) and variants thereof, originating from the epithelium of the mucosal lining of the upper aerodigestive tract (UADT), and adenocarcinomas from associated secretory glands. Carcinomas everywhere in the head and neck spread readily to the lymph nodes of the neck, and this is often the first (and sometimes only) manifestation of the disease at the time of presentation. Head and neck

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_1, © Springer Science+Business Media, LLC 2011

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SCC is strongly associated with certain environmental and lifestyle risk factors, notably tobacco use, smoked and “smokeless”, heavy alcohol consumption, diets poor in antioxidant vitamins and minerals, UV light and occupational exposures to radiation or chemical carcinogens and, increasingly to certain viruses, perhaps sexually transmitted, notably “high-risk” genotypes of the human papillomavirus family (particularly HPV 16 and 18, and particularly when originating in the tonsil and elsewhere in the oropharynx), and some human herpes viruses (HHVs: Epstein-Barr virus with nasopharyngeal carcinoma and HHV-8 with Kaposi sarcoma at all sites): there is a modest inherited susceptibility. SCC of the H&N are frequently aggressive in their biologic behaviour: patients with many of these types of cancer have very destructive disease above the clavicle, develop local (cervical) lymph node metastases early, develop distant metastases over time – even following effective local therapy, and a high proportion have recurrence of the primary lesion and/or develop a second primary neoplasm. This is especially so if risky life-styles continue: UADT cancers ought in fact to be considered systemic diseases; not only is there “field of change” with molecular lesions involving much or all of the regional mucosae, but also damage to the immune system and host defenses generally, and damage to key organs especially the liver. Indeed co-morbidities are common – especially respiratory and cardiovascular – resulting from common risk factors, especially tobacco and alcohol abuse, and poor nutrition. H&N SCC is curable if detected early, usually with some form of surgery. For more advanced lesions, in modern best practice, surgery is usually accompanied by preceding or subsequent radiotherapy, with or without adjuvant chemotherapy. We are now entering an era of individualised biotherapies for many cancers, based on understanding of the precise molecular aberrations within a given neoplasm, and of the patient’s individual genetic polymorphisms, though such approaches have not yet been extensively trialled. The evidence base as it was earlier this decade, with a focus on oral cancer, is exhaustively presented in Shah JP, Johnson NW & Batsakis JG, Oral Cancer, Martin Dunitz London/Thieme New York, 2003.

History Evidence of head and neck malignancies has been found in ancient skulls. The oldest known tumour is contained in a fossil found in East Africa by Leakey that dates back more than 500,000  years. Some historians speculate that a high incidence of nasal cancer may have been present in some ancient populations because of the inhalation of wood smoke

N.W. Johnson and H.K. Amarasinghe

in poorly ventilated huts. In approximately 400 bc, Hippocrates described a common chronic ulcer at the edge of the tongue that he attributed to the presence of sharp teeth rubbing against the tongue: a challenge to differential diagnosis which is still real today! The ancient Indian physician Sushruta described the removal of tumours and developed great skill in plastic surgery, partly from defects created by frequent amputations of the nose and ears for punishments. Modern Western Medicine received its foundation from early Roman medical writings. Little medical advancement was made for head and neck cancers until the advent of anaesthesia and surgical excision in the eleventh century.

Cancer Registries Cancer registries play a vital role in monitoring the incidence of and mortality from cancers. However, the quality of data available in many registries can be far from ideal. Furthermore, many parts of the world produce no data at all, in others (often among the most populous), the data may come from localised, atypical regions. Hospital-based cancer registries naturally gather biased information – those cases which present to hospital only; thus, in many developing countries, cases may not come to attention at all, either because of fear or the inability of poor people to access hospital services. Data may be even more unreliable because in many developing countries follow-up, even of treated cases, is impossible. Death certification is not always compulsory and there is limited international standardisation in the categories for cause of death, let alone calibration of those signing death certificates. Fortunately, many nations have high quality national, often incorporating regional, population-based cancer ­registries, with compulsory reporting of all malignancies. These are guided by, and quality-assured by, both national authorities and the positive influence of the World Health Organisation (WHO), mostly through its constituent body, the International Agency for Research on Cancer headquartered in Lyon, France. Data from all over the world are ­collated and are available from the websites of both these bodies: this includes free access to programmes that allow online interrogation of the databases. Many of the tables and graphs in this chapter have been generated in this way. Within the USA, the SEER website provides similar sophisticated opportunities to registered users (SEER is the Surveillance, Epidemiology and End Results program of the National Cancer Institute. It is based on data from, nowadays, 18 ­population-based registries described at http://seer. cancer.gov/registries/list.html).

1  Epidemiology and Aetiology of Head and Neck Cancers

Why Collect Detailed Epidemiological Data? Cancer epidemiology is a demanding but essential science. Some acquaintance with epidemiological method and data is required by all who participate in cancer care, from politicians, public health officials, hospital managers, individual clinicians in both general and the wide range of specialist practitioners concerned with diagnosis and treatment, palliative carers, nurses, speech and swallowing therapists, dieticians, social workers to spiritual advisors. Descriptive epidemiology provides the fundamental evidence base, but its value is dependent on the accuracy and completeness of the information therein: reliable, sufficiently detailed and safely stored hospital-based information is sine qua non. Increasingly, hospital records contain information on lifestyle and other known or suspected risk factors: the growth of biological “tumour banks” or “tissue banks” from which molecular markers and indeed molecular mechanisms can be researched is encouraging, but needs much more co-­ordinated international action. Population-based registries, as described above, are of even greater value. These permit analytical epidemiology, and thus the ability to address essential questions such as: Why is the incidence of a particular type or site of neoplasm rising or falling over time or in a particular ethnic group or age group? How should this inform government and public health policy? Are existing public awareness and screening campaigns effective and efficient? How do different treatment modalities compare? How does my hospital or my personal clinical practice compare to the national average or

Fig. 1.1  Global scenario of age-standardised cancer incidence and mortality rates per 100,000 population

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world best practice? In respect of the latter, there is an ­ethical imperative for every clinician to keep detailed records, using standardised measures, of the outcomes of his or her care. Guidelines for Care Pathways and “Minimum Data-Sets” to facilitate quality control and recording of outcomes are available: those from the British Association of Head and Neck Oncologists (http://www.bahno.org.uk/docs/) and from the American Head and Neck Society (http://www. headandneckcancer.org/) can be recommended. In many countries, cancer is a notifiable disease and both the registration of all cases, and the provision of information on the patient, on the care provided, and on the outcomes – not just survival rates but information on complications and on ­quality-of-life ­measures – is mandatory.

The Global Scenario of Head and Neck Cancer: Differences by Country Head and neck cancer is the sixth most common type of cancer, representing about 6% of all cases and accounting for an estimated 650,000 new cases and 350,000 cancer deaths worldwide every year [1]. Figure 1.1 compares several H&N cancers with cancers affecting other body sites: age-adjusted global incidence and mortality rates are given for males and females; males predominate in all H&N sites. Head and Neck Cancers are among the Top Ten in the World: We separate nasopharyngeal cancer, because it has a specific aetiology related to EBV infection and dietary

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N.W. Johnson and H.K. Amarasinghe

Fig. 1.2  Simple pie charts of the estimated number of new cases of cancer in the world in 2002, derived from the Globocan 2002 database, divided into the nine most common sites in males (a) and females (b). Note that oral cavity appears in eighth place for males

c­ arcinogens. We then add together those sites with the ­common major risk factors of alcohol, tobacco and diets poor in antioxidants and vitamins, and a minor role for HPVirus – ­collectively termed upper aerodigestive tract cancer (UADT): these sites are oral cavity (including tongue), other pharynx, and larynx. Male incidence/mortality is then 15.2/8.1 and female 4.6/2.4 cases per 100,000 pa. This would rank men approximately sixth in the table; women approximately eighth. Adding nasopharynx pushes head and neck cancer higher up the scale. If oesophagus were to be included as another alcohol and tobacco-related cancer, the rates add to 28.6/18.9 and 10.1/6.8, respectively. These cancers – which might be termed cancers of the mouth, throat and Gullet – then rank second only to lung cancer in men, and fourth after breast, uterine cervix and large bowel in females worldwide (Fig. 1.2a, b). The geographical patterns of oral cancers are indicative of differences in the prevalence of risk factors among countries; tobacco and alcohol consumption, and quality of diet, in particular. Two-thirds of these malignancies occur in developing countries; and a high incidence continues to be observed in the Indian Subcontinent. According to GLOBOCAN 2002, the highest incidence of oral cancer is found in Melanesia (astounding rates of 31.5 per 100,000 in men and 20.2 per 100,000 in women) [2]. In India alone, over 100,000 cases of oral cancer are registered every year. Though men predominate overall, among females, a very high incidence is found throughout southern Asia (8.3 per 100,000). In terms of countries, Sri Lanka has the highest incidence of oral cancer in the South Asia region. Poor access to health services contributes to high mortality. Data extracted from the Cancer Incidence in Five Continents Database for the period 1998–2002 [3] also ­facilitate a global overview. When considering oral and pharyngeal cancer, the annual estimated incidence is around

275,000 cases for oral and 130,300 for pharyngeal cancers excluding nasopharynx: two-thirds of these cases occurring in ­developing countries [2]. There is a wide geographical variation in the incidence of oral cancer, nasopharyngeal cancer, other pharynx and larynx (Table 1.1). For oral cancer, the highest crude rates in the world are found in Melanesia, Hungary, France, Sri Lanka and Croatia [2]. There are marked differences among countries in the same geographical region [4, 5]. The extremely high rates in the relatively small populations of the Melanesian Islands have not been comprehensively researched, but good data from Papua New Guinea (see below) define the importance of areca nut (betel) chewing (called Buai in PNG) and smoking habits as the major risk factors. The World maps reproduced below (Figs. 1.3–1.9), though simplifying data by aggregation to national averages, contain important information. As with the tables, maps are shown for each of the important head and neck sites. It has been apparent for decades that the global picture for head and neck cancer is dominated by the incidence of oral cancer in southern Asia and of oral cavity plus nasopharyngeal cancer in East Asia. In the 1980s, in India, Bangladesh, Pakistan and Sri Lanka, oral cancer was the most common site and accounted for about one-third of all cancers [6–8]. However, this proportion has fallen, mainly due to increased detection of other cancers by more extensive screening programmes and improved techniques [8]. Even within the subcontinent, there are striking differences in incidence rates. The highest rate for tongue and mouth is reported for men living in South Karachi, Pakistan; the second highest in Trivandrum city, Kerala, India. Extremely high rates for women are seen in the Tamil community in Malaysia – higher even than in Tamil Nadu itself: UADT sites in Indian females in Peninsular Malaysia are the second most common cancer, behind breast and above uterine cervix [9].

Table 1.1  World standardised incidence rates per 100,000 for H&N cancers. Data derived from the Globacan 2002 database: anatomic descriptors derived therefrom [2] Oral cavity Nasopharynx Other pharynx Larynx Country Male Female Male Female Male Female Male Female World More developed Less developed Eastern Africa Middle Africa Northern Africa Southern Africa Western Africa Caribbean Central America South America Northern America Eastern Asia Southeastern Asia South Central Asia Western Asia Central and eastern Europe Northern Europe Southern Europe Western Europe Australia New Zealand Melanesia Micronesia Polynesia

Fig. 1.3  Mouth Cancer Deaths, IARC 2002 International Classification of Diseases-10 codes: C00-C14. http://www. worldmapper.org/display_extra. php?selected=419. Accessed January 2010. These two maps (shown only for males here) distort countries on the basis of the number of deaths by mouth and pharynx cancer (a), and the number of smokers (b). They show that the public health burden is borne by Eastern Europe, Central and Eastern Asia and South Asia. China is the major storehouse of tobaccorelated morbidity and mortality in the world, a nation where more than half the population continues to smoke. Yemen, Indonesia and Mongolia = Armenia, followed by Kenya are the top five-ranked countries for smoking prevalence, at 77%, 69%, 68% and 67%, respectively. Territory size shows the proportion of men who smoke and live there

6.3 7.9 5.7 5.9 4.4 3.1 11.1 2.5 5.6 2.5 6.1 7.8 1.5 3.6 12.7 3.7 8.6 5.3 9.2 11.3 11.1 5.6 31.5 4.4 5.2

3.2 2.4 3.5 4.8 2.2 1.5 3.1 1.3 2.3 1.4 1.8 3.3 1.0 2.5 8.4 2.3 1.6 2.6 2.0 2.7 4.7 3.3 20.2 2.7 0.7

1.9 0.7 2.4 2.3 0.8 2.7 1.6 1.7 0.6 0.4 0.3 0.6 3.3 5.8 0.7 1.6 0.6 0.4 1.1 0.8 0.8 0.6 0.3 7.1 3.9

0.8 0.2 1.0 0.9 0.2 1.1 0.4 0.7 0.2 0.2 0.1 0.2 1.5 2.1 0.3 0.6 0.2 0.2 0.5 0.2 0.3 0.3 0.2 2.5 1.7

3.8 5.1 3.4 1.6 1.2 1.0 2.0 0.8 4.3 1.7 3.5 4.2 0.7 2.2 8.8 0.7 5.4 2.6 5.3 9.7 3.4 3.6 1.8 1.6 0.7

0.8 0.8 0.8 0.5 1.1 0.2 0.4 0.2 1.2 0.6 0.7 0.9 0.1 0.7 2.0 0.4 0.5 0.7 0.5 1.7 0.9 0.4 0.7 0.6 0.0

5.1 6.9 4.3 3.5 2.6 4.0 6.5 2.0 7.3 5.0 7.2 5.8 2.0 3.7 6.9 7.2 9.2 4.3 10.9 7.2 3.9 2.2 3.2 3.7 0.0

0.6 0.7 0.6 0.7 0.8 0.6 0.9 0.3 1.2 0.4 0.7 1.2 0.3 0.5 1.0 1.2 0.4 0.7 0.7 0.8 0.5 0.4 0.9 0.0 0.0

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Fig. 1.4  Incidence (a) and mortality (b) rates for oral cavity cancer in males, in quintiles, by country. A quick comparison of these maps makes a number of points. The “traditional” high incidence areas of central Asia and the Indian sub-continent stand out: much of this is due to betel quid use, with or without smokeless tobacco, plus smoking, sometimes alcohol abuse, and poor diet. Note that parts of both Western and Eastern Europe remain in the top quintile – see text. The African data are not particularly robust. Australia shows a high incidence, due to ultraviolet light-induced lip cancer in a fair-skinned population:

N.W. Johnson and H.K. Amarasinghe

­ ortality rates are not comparably high because lip cancer is comparam tively easily treated. Eastern Europe and the former Soviet republics have high mortality, partly related to low socio-economic status, limited treatment facilities and the fact that many patients have substantial ­co-morbidities. As already mentioned, Papua New Guinea and surrounding Melanesian islands of the Western Pacific are in the top quintile both in incidence and mortality: indeed Melanesia has the highest recorded rates in the world at the beginning of this millennium – ­associated with chewing of areca nut and tobacco use

1  Epidemiology and Aetiology of Head and Neck Cancers

Fig.  1.5  Similar explanations relate to the national incidence (a) and mortality (b) data for women. Note the serious situation in the Indian Subcontinent and parts of SE Asia. In parts of India, oral cancer is the

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leading cancer among women, because of heavy use of betel quids. Indeed emigrant Tamil women working on rubber and palm oil estates in Malaysia have among the highest rates, by population group, in the world

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Fig. 1.6  Rates of laryngeal cancer largely reflect smoking rates around the globe, with the surprising exceptions of China and Japan who have comparatively low incidence (a) and mortality (b), in spite of male smoking prevalence being 50% or above: however as noted earlier

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Japanese rates are on the rise. The proportionately higher death rate in Eastern Europe, Russia and the former Soviet Republics is again related to late stage at diagnosis and high co-morbidities associated with low socio-economic status and difficulties with access to care

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Fig. 1.7  (a and b) Because smoking is far less prevalent in women than men in most societies, the laryngeal cancer rates are low worldwide, and little can be read into this aspect of “geographical pathology”

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Fig.  1.8  Risk factors for nasopharyngeal cancer are comparatively well understood. It is a biologically distinct disease, driven by EpsteinBarr virus, in subjects with genetic susceptibility, compounded by toxins in particular cultural dietary practices. Both incidence (a) and

N.W. Johnson and H.K. Amarasinghe

mortality (b) rates are historically high in North Africa and in China – particularly Guangdong Province, the Hong Kong SAR and emigrant communities there from

1  Epidemiology and Aetiology of Head and Neck Cancers

Fig. 1.9  (a and b) Female rates for NPC are lower than for men, but show the same geographical distribution

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More than 180,000 cases of oral cancer occur every year in South and South-East Asia alone, with poor prospect of survival: about 90% of these cases are attributable to smoking and chewing habits [7]. It is encouraging that overall rates in India are showing a decreasing trend in successive birth cohorts, declining trends were observed for mouth (ICD10 C03–C06) and tongue (C01–C02) cancers among females and tongue cancers among males between 1982 and 2000 [10]. However, there is growing concern that commercial areca nut and tobacco products will contribute to future rises in the incidence of oral submucous fibrosis and of subsequent oral cancer [11]. Data from Japan show a dramatic increase in oral and pharyngeal cancer incidence (ICD10 C01–C14) for both sexes; there is a 4.4-fold increase for males and 3.8-fold increase for females in the total numbers between 1965 and 1999 – noted from the data retrieved from Osaka Cancer Registry’s database [12]. There is also an upward trend for both males and females in Australia and among the nonMaori population in New Zealand. Lip cancer in fair-skinned populations, particularly due to ultraviolet light, is a growing problem [13]. In Europe, Hungary has the highest incidence and mortality of oral and pharyngeal cancer for both sexes [14]. Between 1984 and 1994, the Hungarian mortality rates for oral cancers rose by 83.5 and 72.3% in males and females, respectively. Trends in the mortality rate among Italian and French males peaked in the 1980s and have decreased after 1990 [15]. However, some persisting upward trends were registered for Belgium, Denmark, Greece, Portugal, and Scotland [16]. In the USA, the estimated number of incident cancer cases for tongue, mouth and other oral cavity in 2008 was 15,250 cases for men and 7,650 for women; for the pharynx, the number of incident cases for men is 10,060 and 2,350 for women (3% of all cancer cases in men). For cancer of the larynx, 12,250 incident cases were estimated, of which 9,680 were men. In the USA, the mortality rates per 100,000 population pa for cancer of the oral cavity and pharynx for men was 5.61 in 1990 and 3.98 in 2004, the absolute decrease being 1.63 per 100,000, contributing to a 3% reduction in mortality of all sites. For women, the decrease across the same period was 0.56 contributing to a 2.5% reduction of all sites [12]. The incidence rates of cancers of the oral cavity and pharynx-throat were stable or declining for men and women in most age groups during the period 1973–2003 in the USA, probably related to changes in tobacco and alcohol consumption. This is a highly pleasing situation, common to many countries with advanced care facilities but not reflected in most of the high incidence countries elsewhere in the world. Furthermore, as described below, black citizens of the USA fare comparatively badly. Cancer of the larynx has always been a serious public health problem in nations with high smoking prevalence, and

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this remains a disaster in China and eastern Europe and the former Soviet Republics. Differences among selected countries are shown in detail in the time and birth cohort trends reproduced below. For cancers of the oropharynx and tonsils, the highest combined rate is currently seen in France and for laryngeal cancer, it was Spain. For hypopharyngeal cancer specifically, the highest rate in men was in France. For women, the highest ASR(W) for mouth and tongue specifically was in Pakistan, almost the same as that for men [17].

Differences by Sex As already noted, worldwide, the incidence of head and neck cancers overall is higher for males than females. According to the International Agency for Research on Cancer [2], the agespecific incidence of “oral cavity”, plus “nasopharynx” plus “other pharynx” cancers totalled 12 per 100,000 population for males in 2002 and 4.8 for females (see Table 1.1). This may be because of their greater indulgence in the most important risk factors, such as heavy alcohol and tobacco consumption for intra-oral cancer and sunlight for lip cancer in those who work outdoors. However, oral cancer in females is increasing in some parts of the world. For instance, a study from Argentina showed the male/female ratio to be 1.24:1 for the period 1992– 2000 compared to 7.1:1 for the 1950–1970 period [18]. The incidence of tongue and other intra-oral cancers for women can be greater than or equal to that for men in high incidence areas such as India, where betel quid/areca nut chewing (and sometimes smoking) are common among women, although this varies considerably from region to region. Within Europe, the incidence of oral cavity and pharyngeal cancers (C00–14) among males in the most recent period varied substantially between 5.9 (Finland) and 32 (France) per 100,000 pa [19]. Incidence rates among females were highest in northern and western Europe but were consistently lower than those for males. The male-to-female ratio decreased during the last 10  years and recently varied between 1.5 and 2.5 in northern Europe and 7.7 in Lithuania. Between 1990 and 1999, the UK incidence rates for oral ­cancers rose in males of all ages from 6.5 to 8.3 per 100,000 (an increase of 18%) and in females from 2.6 to 3.6 per 100,000 (an increase of 30%) [20]. In the USA, the death rate due to cancer of the oral cavity and pharynx per 100,000 population in 2005 was 3.8 for males and 1.4 for females, down from 6.9 to 2.3, respectively in 1975. This substantial improvement is not reflected in most of the rest of the world. Apart from the traditional risk factors, it has been suggested that oestrogen deficiency may influence susceptibility to oral cancer in women: Significantly, younger mean age at

1  Epidemiology and Aetiology of Head and Neck Cancers

menopause and higher rates of hysterectomy may influence the higher rates of oral cancer seen among younger females [21]. Data presented in this chapter are, whenever possible, separated by sex.

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people around them. Passive smoking by children can increase the risks of asthma, cot deaths and chest infections. The prevalence of smoking increased dramatically during the world wars, mainly due to the policy of providing free cigarettes to allied troops as a “morale boosting” exercise. The Cancer Council, 2006.

Ethnic Variations Variations by ethnicity are largely due to the social and cultural practices, and the influence of dietary and genetic factors, though the latter are less well quantified. Variations in outcome are also contributed to by differences in access to healthcare. Where cultural practices represent risk factors, their continuation by immigrants from high incidence regions to other parts of the world results in comparatively high cancer incidence in immigrant communities. This can also affect the sub-sites of oral cancer most commonly affected, as shown in a recent study from California [22]. The highest ageadjusted oral cancer rates in the USA are found among nonHispanic black men (4.86/100,000) followed by non-Hispanic black women (4.71/100,000), with Asian and Hispanic populations showing intermediate incidence rates compared with white (Caucasian) ethnic groups. Tongue cancer was the most common type of oral cancer among every ethnicity. Asians were more likely to develop their malignancy in the buccal mucosa, a reflection of continuing areca and tobacco chewing habits. Another study showed that American Indians and Alaskan Natives overall had significantly lower incidence rates than non-Hispanic whites [23]. Several studies from the USA have demonstrated that black patients with oral cancer have poorer overall and disease-specific survival than whites, mainly because of their comparatively poor access to health care [24, 25]. This is especially concerning because the incidence of oral plus pharyngeal cancer for black men in the USA is so high, and is the sixth most common site for malignant disease among this group [26]. The age-adjusted incidence rate for oral and pharyngeal cancers is higher for South Asians than for other residents in England, particularly among females [27]. Interestingly, this study showed that British South Asian males have significantly better survival than their non-South Asian peers in the south east of England, possibly a reflection of the more indolent progress of tobacco/areca nut-induced lesions [27]. Worldwide there are four times more men who smoke than women. In 2002, there were 941 million male smokers, which was 43% of all men aged over 15 years old. The largest population of male smokers lives in China – where men are more likely to smoke than not to smoke. Even Puerto Rico and Sweden, with the lowest percentages of men who smoke still have 17% who are smokers (Fig. 1.3a, b). When smoking is widespread, smokers not only just ­damage their own health, but also collectively damage the health of

Age Distributions Oral cancer is usually a disease that occurs in males after the fifth decade of life. The mean age at presentation is in the fifth and early sixth decades in Asian populations compared with the seventh and eighth decades in the North American population [28–33]. Statistics in the USA for 2001–2005 show that the median age at diagnosis for cancer of the oral cavity and pharynx was 62 years [34]. Several studies suggest that 4–6% of oral cancers now occur at ages younger than 40  years [35]. An alarming increase in incidence of oral cancers among younger people has been reported from many parts of the world [36–39], a trend that appears to be continuing. There was a significant increase in the incidence of cancers in the tongue and tonsil among 20–40 year olds in the USA between 1973 and 2001 [40]. In Germany, Czechoslovakia and Hungary, there has been an almost tenfold rise in mortality from oral cancer in men aged 35–44 [41], within one generation. Robinson and Macfarlane showed a dramatic increase in incidence rates for younger males in Scotland from the 1980s to the 1990s [42]. In the high prevalence areas of the world, in many cases patients are less than 40 years old, probably owing to heavy lifetime use of various forms of tobacco, although some recent Indian data have not shown this [43]. It is also clear that a number of cases of squamous cell carcinoma occur in both young and old patients, often in the absence of traditional alcohol and tobacco risk factors, and in which the disease may pursue a particular aggressive course. A study conducted in Southern England concluded that a substantial proportion of cases of younger people diagnosed with oral cancer occur in the absence of known risk factors [44]. This, together with the relatively short duration of exposure in users suggests that factors other than tobacco and alcohol are implicated in the development of oral cancer in a significant minority of cases. Diets poor in fresh fruits and vegetables were identified as conferring significant risk. HPV infections may also be relevant in a proportion of these cases. It is also suggested that greater attention should be paid to familial antecedents of malignant neoplasms in younger patients with oral cancer [45]. Age distribution curves for the major head and neck cancer sites are given for deliberately selected countries in Figs. 1.10–1.15.

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Fig. 1.10  Male age-specific incidence curves for mouth and pharynx for selected countries. All UADT cancers show a similar distribution. Most cases occur in the fifth to seventh decades of life, presumably because decades of exposure to tobacco, alcohol and poor nutrition take time to synergise with other agents in triggering malignant transformation – or in allowing this to survive the host response!! There are, nevertheless, a significant minority of cases appearing in the third and fourth decades of life: these attract much interest as, although associations with early commencement of smoking, and with unsafe alcohol use can be demonstrated, a substantial minority of cases arise without exposure to traditional risk factors: here dietary inadequacies and HPV infection are thought to be important, as may inherited predisposition. In the high incidence age bands there is a ~40–100-fold difference in incidence with, among the countries selected here, disturbingly high rates in NW France, Brazil and South India. Note the much worse situation in American blacks cf. whites, explained by a mixture of risk-factor and socio-economic reasons. Finland does comparatively well – not surprising in view of that nation’s success in reducing the prevalence of smoking, though alcohol abuse remains a social problem. What is surprising are the low rates recorded for Shanghai, in spite of high smoking prevalence in this large city. China is in the early stages of developing a comprehensive, nation-wide Cancer registry system and caution is necessary in interpreting some of the current data

Mortality Rates and Trends over Time Table  1.2 gives mortality data again extracted from the Cancer Incidence in Five Continents Database for the period 1998–2002 [3], for comparison with the incidence data in Table  1.1. Trends of age-standardised (world population) mortality rates for the head and neck cancer sites of interest,

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Fig. 1.11  Rates for females are lower and international differences are less marked. Women in South India stand out – related to use of betel quid and tobacco, together with low SES

within selected countries over the past three to six decades, are presented in Figs. 1.16–1.21. Current male death rates for oral and pharyngeal cancer around the world are seen vividly in Fig. 1.16. There was a steady rise in oral cancer mortality in men from the 1950s to late 1980s in most Western European countries [46], but this trend has since declined, in France, China and Hong Kong, which had exceedingly high rates in the past. Unfortunately, in most countries in central and eastern Europe, oral cancer mortality in men has continued to rise, reaching exceedingly high rates in Hungary, Slovakia, Slovenia and the Russian Federation. Hungary, Ukraine, Estonia and Bulgaria show more than a 100% increase in mortality rates for men during the 20-year period up to the turn of the Millennium. Even though the rates of oral cancer are comparatively low among women, there is a steady increase in some countries in Europe (notably Hungary, Belgium, Denmark and Slovakia). Hungary also shows a 98% increase in mortality rates for women Fig.  1.17. These disturbing trends are thought to relate to high drinking and smoking patterns in these societies, together with poor diet in lower socio-economic groups. Trends for laryngeal cancer reflect continuing high rates of tobacco consumption in many societies (Figs. 1.18 and 1.19). Trends for naso-pharyngeal cancer, both good and bad, are shown for high-incidence countries (Figs. 1.20 and 1.21).

1  Epidemiology and Aetiology of Head and Neck Cancers

Fig. 1.12  Many of the differences between populations are likely to be explained by smoking and other traditional risk factors. Serious public health challenges exist in the Brazilian example. Poland and the Russian example are consistent with the major concerns we have for Eastern Europe, Russia and the former Soviet republics as a whole. Blacks do poorly in the USA. Finland provides encouragement: indeed this was the first country in the world to reach the WHO target for the year 2000 of having less than 20% of the adult population smoking. Japan and China remain enigmas

Mortality Trends by Birth Cohort This is a valuable way for determining time trends. Cases of particular cancers are transformed back, in 5-year age groups, to the date of birth of the affected individuals. Curves for particularly instructive countries are given below. In general these show that for most UADT cancers, in most developed countries, rates fell in the latter part of the nineteenth and the first part of the twentieth centuries. This has been continued in, for example, the USA (Fig. 1.22) and the UK (Fig. 1.23). However in Hungary (Fig. 1.24: and the same is true for most of eastern Europe, Russia and the former Soviet republics), those born in the first half of the twentieth century showed alarming rises in death rates. All of these birth cohorts have now passed on, or they are in the highest risk age groups: in these countries, we have thus seen a growing epidemic of UADT cancer. The curves provide limited hope that Hungary at least, may be showing some control in younger people.

15

Fig. 1.13  Although at first glance the spread for women looks larger, the rates are much lower than for men. Again, however, Brazil and American blacks stand out

France (Fig.  1.25) is an interesting case: again the data show that this nation has “turned the corner” with a rise, and now a downturn for cohorts born since the end of the Second World War. The SEER programme in the USA has reported an overall fall in the mortality from oral and pharyngeal cancer, between 1975 and 2004, of 1.87% per annum Table 1.3. Table 1.3 shows a fall in all mortality rates for oral and pharyngeal cancer in the USA between 1975 and 2004. There is a considerable fall in mortality among both white and black women under 65  years of age (APC of −3.12 and −3.21, respectively). Furthermore, the SEER data show higher 5-year relative survival rates for whites (61.8%) and blacks (39.5%), who were diagnosed during the period 1995–2001, than rates for those who were diagnosed during the period 1974–1976 (when rates for whites and blacks were 55 and 36.3%, respectively) [47]. The 5-year survival rates in the SEER Registries range from a high of 72.1% for white women in Utah to a low of 24.8% for black men in metropolitan Atlanta. These striking differences are likely to be explained by a number of factors including socio-­ economic condition, age, stage at diagnosis, continued presence or absence of environmental risk factors and access to

16

Fig. 1.14  NPC is a distinct disease. These countries have been chosen to reflect the differences by population. As mentioned in the legend to the cancer map, southern Chinese men are particularly susceptible: hence the alarming data from Hong Kong and to a lesser extent from Shanghai. Although the data are fragmentary, the markedly higher rates in Chinese Hawaiians than other racial groups there is consistent with the ethnic bias

h­ ospital services. African-American patients have consistently poorer survival outcomes [48]. A study in Mumbai, India, indicated a decreasing trend in oral cancer incidence among Indian men, which it was suggested may be due to a decrease in the use of betel quid/pan and associated oral smokeless tobaccos over this period [49]. However, there continues to be a high prevalence of smokeless tobacco use among young adult men and women, especially in the form of Pan Parag/Gutka-type products, and cigarette smoking is increasing. Overall, UADT cancers are not likely to decrease. Population-based survival rates around the world show little evidence of improvement over recent decades, despite vast improvements in treatment modalities. Cure rates and survival rates have improved with advances in surgical and other techniques in highly specialised, high-volume treatment institutions. Regrettably, such highly expert ­management is not yet uniformly available and it will be many more decades before these results are reflected in ­population trends.

N.W. Johnson and H.K. Amarasinghe

Fig. 1.15  The highest rates of NPC in women are again in Chinese – though only a tenth of those in males

Aetiology of Head and Neck Cancer The majority of oral SCC are related to tobacco in various forms, betel quid chewing, heavy alcohol drinking and dietary micronutrient deficiency. In the developing world, tobacco and areca nut, used either alone or in combination, accounts for the vast majority of oral cancers and oral potentially malignant disorders (OPMD) [50]. The WHO has classified areca nut, a common component of many different chewing habits, as carcinogenic to humans [51]. UV radiation is relevant to lip cancer and there is an increasing evidence for a role for “high risk” genotypes of the HPV family, especially for tonsillar and other oropharyngeal sites.

Betel Quid A betel quid generally contains betel leaf, areca nut and slaked lime, and may contain tobacco. Other substances, particularly spices, including cardamom, saffron, cloves, aniseed, turmeric, mustard or sweeteners, are added according to local preference [51].

1  Epidemiology and Aetiology of Head and Neck Cancers

17

Table 1.2  World standardised global mortality rates per 100,000 for H&N cancers. Data derived from the Globocan 2002 database: anatomic descriptors derived therefrom [2] Oral cavity Nasopharynx Other pharynx Larynx Country Male Female Male Female Male Female Male Female World 2.7 1.5 1.2 0.5 2.5 0.5 2.9 0.4 More developed 2.7 0.7 0.3 0.1 2.5 0.4 3.3 0.3 Less developed 3.0 1.9 1.6 0.7 2.5 0.6 2.7 0.4 Eastern Africa 3.5 2.9 1.7 0.7 1.2 0.4 2.9 0.6 Middle Africa 2.7 1.3 0.6 0.2 0.9 0.8 2.1 0.7 Northern Africa 1.9 0.9 2.0 0.8 0.7 0.1 3.3 0.5 Southern Africa 6.3 1.7 1.1 0.3 1.5 0.3 4.2 0.5 Western Africa 1.5 0.8 1.2 0.5 0.6 0.2 1.6 0.3 Caribbean 2.7 1.0 0.5 0.1 2.4 0.7 4.5 0.8 Central America 1.0 0.5 0.2 0.1 1.0 0.3 2.5 0.4 South America 2.3 0.8 0.2 0.1 2.3 0.4 3.9 0.6 Northern America 1.4 0.6 0.3 0.1 1.3 0.4 1.7 0.3 Eastern Asia 0.7 0.4 2.1 0.9 0.3 0.1 1.0 0.2 Southeastern Asia 1.9 1.3 3.8 1.3 1.7 0.5 2.3 0.3 South Central Asia 7.0 4.6 0.5 0.2 6.7 1.5 4.5 0.6 Western Asia 1.8 1.1 1.0 0.4 0.5 0.3 4.4 0.7 Central and eastern Europe 5.1 0.7 0.4 0.1 3.9 0.3 6.8 0.3 Northern Europe 1.9 0.8 0.2 0.1 1.3 0.3 1.6 0.3 Southern Europe 2.7 0.6 0.4 0.1 2.5 0.3 4.8 0.2 Western Europe 2.9 0.8 0.2 0.1 4.0 0.6 2.9 0.3 Australia 1.8 0.9 0.3 0.1 1.5 0.3 1.6 0.2 New Zealand 1.3 1.0 0.3 0.1 1.0 0.2 0.9 0.2 Melanesia 17.3 11.7 0.2 0.2 1.4 0.5 1.9 0.7 Micronesia 2.4 1.5 4.9 1.6 1.2 0.5 2.1 0.0 Polynesia 2.8 0.3 2.5 1.1 0.6 0.0 0.0 0.0 Note again the stand-out rates for Melanesia! All head and neck cancers are highly morbid and lethal. Taking oral cavity, as defined in these tables, Death to Registration ratios (D/R) are 0.34 for males and little better at 0.29 for females in the more-developed countries: an appalling 0.52 and 0.54, respectively, in less-developed countries where they come late to diagnosis, where there are significant co-morbidities and where quality care is less available. This means that in most of the world, more than half of the individuals diagnosed with an oral cancer die of their disease

Fig. 1.16  Trends in mortality over time are important to track, and to understand. Hungary is a disaster, though hopefully the rise has been arrested. Russia remains a concern. France demonstrates what can be achieved, overall, in spite of the concerns shown by the Calvados registry data above. The overall downward trend in the other countries illustrated is encouraging

18 Fig. 1.17  Although only approximately a tenth of the male rate, Hungarian females remain a challenge

Fig. 1.18  Another success demonstrated for France. Have Russia and Hungary genuinely turned the corner?

N.W. Johnson and H.K. Amarasinghe

1  Epidemiology and Aetiology of Head and Neck Cancers Fig. 1.19  This is a “noisy” curve because of the comparatively low mortality rates in women. Worryingly, but not surprisingly, it suggests an upward trend in Hungary

Fig. 1.20  One hopes the successes in Hong Kong can be replicated in other high risk groups

19

20

N.W. Johnson and H.K. Amarasinghe

Fig. 1.21  From a lower initial base, Hong Kong women share this success story

Fig. 1.22  Birth-cohort curves of the mortality rates for lip, oral cavity and pharyngeal cancers for USA males (a) and females (b)

1  Epidemiology and Aetiology of Head and Neck Cancers

21

Fig 1.23  Birth-cohort curves of the mortality rates for lip, oral cavity and pharyngeal cancers for males (a) and females (b) England and Wales

Fig. 1.24  Birth-cohort curves of the mortality rates for lip, oral cavity and pharyngeal cancers for males (a) and females (b), and for laryngeal cancer (c, d) in Hungary. The challenge for Hungary, apparent in other

curves, is confirmed here. Males born in the first half of the twentieth century had rising rates or death from oral and pharyngeal cancer. There are indications that those born after 1950 may be less at risk

Fig. 1.24  (continued)

Fig. 1.25  Birth-cohort curves of the mortality rates for lip, oral cavity and pharyngeal cancers for males (a) and females (b), and for laryngeal cancer (c, d) in France. Birth cohort curves are instructive. For males born in the nineteenth century and the first few decades of the twentieth

century, death rates from oral and pharyngeal cancer were extremely high. Those born from around 1940 and later are generating the national average downward trends seen above

1  Epidemiology and Aetiology of Head and Neck Cancers

23

Fig. 1.25  (continued)

Table 1.3  Mortality trends (APC) for oral and pharyngeal cancer in the USA between 1975 and 2004, by race and sex. (SEER Cancer Statistics Review, 1975–2004) [34] All races Whites Blacks Total Males Females Total Males Females Total Males Females All ages −1.87a −2.12a −1.63a −1.87a −2.15a −1.62a a a a a a Under 65 −2.41 −2.24 −3.06 −2.38 −2.18 −3.12a 65 and over −1.45a −1.99a −0.75a −1.53a −2.14a −0.79a APC annual percentage change a  The annual percentage change in rate is statistically significantly different from zero (p < 0.05).

−1.94a −2.88a −0.46a

−1.91a −2.80a −0.59a

−1.71a −3.21a −0.26

Betel Leaf

Betel Inflorescence

The leaves of the Piper betel vine (a member of the pepper family) contain betel oil, a volatile liquid, which contains several phenols including hydroxychavicol, eugenol, betel phenol and chavicol. These compounds may, to some extent, be protective, sharing some of the antioxidant properties of many plant polyphenols. Vitamin C, a large amount of carotene and 36 trace elements have also been reported in the betel leaf, clearly beneficial micronutrients [52].

Apart from the leaf, other parts of the vine such as stem, inflorescence (the flowers or pods) or catkins are also ­consumed with areca nut. Consumption of the inflorescence is common in Melanesia and parts of Taiwan, and in China, and it is mostly added to the quid for its aromatic flavour [51]. Betel inflorescence contains a high concentration of phenolic compounds including hydroxychavicol, eugenol, isoeugenol, eugenol methyl ester and safrole. Safrole itself, a major

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p­ henolic compound, is classified as a weak carcinogen in rats and is banned as a food and cosmetic additive by the FDA in the USA, inter alia, however, there is no direct evidence for its carcinogenicity in man.

N.W. Johnson and H.K. Amarasinghe

exported worldwide where they are used by old and new habitués. The most common are gutka and pan masala. Gutka is a dry, ­relatively non-perishable commercial preparation containing areca nut, slaked lime, catechu, condiments and powdered tobacco. The same mixture without tobacco is called pan masala [57].

Areca Nut Areca nut is the seed of the fruit of the oriental palm Areca catechu. It is the basic ingredient of a variety of widely used chewed products. The consumption of areca nut is ­indigenous to India, Sri Lanka, Bangladesh, Myanmar, Taiwan and numerous islands in the South Pacific. It is also popular in parts of Thailand, Indonesia, Malaysia, Cambodia, Vietnam, Philippines, Laos and China, and in emigrant communities from these countries. It is believed that Areca catechu may be native to Sri Lanka, West Malaysia and Melanesia. Areca nut is used as a masticatory substance by approximately 600 million people worldwide. It is estimated that 10–20% of the world’s population chew areca nut in some form, often mixed in betel quid (pan) [51]. The major constituents of the nut are carbohydrates, fat, proteins, fibre, polyphenols (flavonols and tannins), alkaloids and mineral matter. Among the chemical constituents, alkaloids are the most important chemical. The nut has been shown to contain at least six related alkaloids, of which four (arecoline, arecaidine, guvacine and guacoline) have been conclusively identified [53]. Nitrosamine derivatives from each of the four major arecal alkaloids are produced by nitrosation of the alkaloids in dried-stored nuts, in the mouth and especially in the acid conditions found in the stomach, in the presence of nitric oxide generated by bacterial action. Two of these derivatives are accepted as carcinogenic in animal studies, especially MNPN (methylnitrosaminoproprionitrile). Endogenous nitrosation is significantly higher in subjects with poor oral hygiene as determined by volumes of dental plaque [54]. This implies that, on the basis of the availability of substrates from both areca nut and tobacco, there is a more extensive formation of nitrosamine in subjects with poor oral hygiene if they also chew tobacco [55]. Moreover direct evidence that reactive oxygen species, such as the hydroxyl radical (HO), are generated in the oral cavity due to auto-oxidation of polyphenols contained in areca nut and enhancement by the alkaline pH from slaked lime has been reported [51, 56].

Areca Nut-Based Industrial Packaged Products A variety of packaged areca products are now available. These are mostly manufactured in India and Pakistan, and

Damage to Oral Soft Tissues from the Chewing of Areca Nut and Related Products (a) Lichenoid Lesions Areca-induced lichenoid lesions, mainly on buccal mucosa and tongue, are recognised. This is considered to be a type IV contact hypersensitivity-type lesion that resembles oral lichen planus clinically [58]. (b) Betel Chewer’s Mucosa This condition was first described by Mehta et al. (1971), and is characterized by a brownish-red discoloration of the oral mucosa. It is often accompanied by encrustation of the affected mucosa with quid particles, which are not easily removed, and with a tendency for desquamation and peeling. Both chemical and traumatic effects of the betel quid are likely on the oral mucosa. The presence of tobacco in the quid is not essential for the development of chewer’s mucosa [58]. (c) Oral Leukoplakia A case control study conducted in Taiwan, where areca is chewed without tobacco, found the odds ratio for developing leukoplakia to be 7.43 (95% CI 1.94–156.27) for areca nut chewers. These authors demonstrated that the cessation of areca chewing resulted in regression of 62% of leukoplakias [59]. (d) Oral Submucous Fibrosis (OSF) It is now accepted that chewing areca is the single most important etiological factor for the development of OSF [60], although the pathogenesis is not fully understood. In vitro studies have shown that areca nut alkaloids such as arecoline and its hydrolysed product arecaidine can stimulate cultured fibroblasts to proliferate and synthesise collagen. In addition, flavonoids from the nut have been shown to enhance the cross-linking of collagen, thereby increasing its resistance to degradation by collagenases, as part of normal tissue homeostasis. The copper content of areca nut is high and the possible role of copper as a mediator of fibrosis is supported by the demonstration of up-regulation of lysyl oxidase in OSF biopsies [61]. (e) Oral squamous cell carcinoma (OSSC) Historical evidence dating back nearly a century indicates that areca nut is involved in the development of OSCC. Subsequently, many case–control studies [62, 63] have confirmed that betel quid chewing increases the

1  Epidemiology and Aetiology of Head and Neck Cancers

risk of developing OSSC, especially when the quid ­contains tobacco. A South African study found that 68% of cheek cancer and 84% of tongue cancers developed in subjects consuming areca without tobacco [64]. A large number of animal studies have confirmed that areca products and derivatives such as arecoline and arecaderived nitrosamines have the ability to induce neoplastic changes in experimental models, and the IARC has now formally designated areca and betel quids without tobacco as carcinogenic to man [51].

Slaked Lime Slake lime (calcium hydroxide) is added to betel quids in most of South Asia. In coastal areas of Sri Lanka and the Pacific, it is obtained by heating sea shells or harvested from corals. In inland areas, it is quarried from limestone. When added to betel quids, it causes erosions of oral mucous membranes, which facilitate penetration of betel-quid carcinogens through the mucosa.

Smokeless or Chewing Tobacco Tobacco is often added to the quid mixture. Edible tobacco in the Indian subcontinent is prepared from sun-dried and partly fermented, coarsely cut leaves of Nicotiana rustica and Nicotiana tabacum without further processing. Chewing tobacco results in a local exposure of oral mucosa to at least 16 carcinogens, including tobacco-specific nitrosamines (TSNA) and polycyclic aromatic hydrocarbons (PAH) [65]. Unusually high levels of carcinogenic TSNAs (e.g. NNN – N-nitrosonornicotine, and NNK) were reported in saliva of oral snuff users in the Sudan [66] and tobacco chewers in India [67]. NNK is a potent carcinogen and human buccal epithelial cells (in culture) have been shown to be to metabolise NNK: The formation of macromolecular DNA adducts following NNK metabolism is correlated with carcinogenesis in animal models [68]. Betel chewing also releases large amounts of a reactive oxygen species (ROS), especially while the betel quid is actually present. Both TSNA and ROS are major genotoxic agents involved in chewing tobacco-associated oral cancer [51]. Clear dose–response relationships between quid use and the risk of oral cancer and of potentially malignant oral disorders have been demonstrated in many epidemiological studies. Most forms of oral smokeless tobacco – oral snuff – ­consumed in Scandinavia and in North America are not fluecured, and contain relatively low amounts of TSNs. Although

25

the topic is controversial, many of these products are not highly carcinogenic and it has even been suggested that they have a role as nicotine replacement products in achieving smoking cessation [69]. It is, however, important to remember that there is no such thing as safe tobacco: most smokeless tobaccos have high levels of nicotine and are addictive; indeed, there is an evidence that they can be initiators of smoking [70]. Furthermore, they have significant cardiovascular effects [71] and certainly produce oral mucosal lesions and local damage to the periodontium [72].

Contaminants Areca nut can be contaminated with fungi such as Aspergillus flavus, A. niger and Rhizopus spp. Almost 40% of samples of areca nut from India analysed using thin layer chromatography contained aflatoxins [73]. These are established carcinogens.

Tobacco Smoking Tobacco is identified as the leading preventable cause of premature death worldwide. It is estimated that 4.9 million people died of tobacco-related illness in 2000, and by 2020, it is expected that this figure will rise to 10 million deaths per year, of which 70% will be in developing countries [68]. Tobacco is a major independent risk factor for the development of oral and pharyngeal cancer and other malignancies of the upper aerodigestive tract. Tobacco is consumed in different ways as a form of smoking: cigarettes, cigar, beedi, reverse smoking and smokeless tobacco like oral snuff or in moist pouches. Tobacco smoke contains more than 60 carcinogenic combustion products. In particular, NNK, NNN and polycyclic aromatic hydrocarbons (PAHs) have been causally linked to UADT cancer. The activity of carcinogens is generally exerted through DNA adducts [74, 75]. Both tobacco smoking and quid chewing cause oxidative stress to tissues, that is, the sustained presence of reactive oxygen species (ROS), which initiate free radical reactions. ROS can damage proteins, lipids, carbohydrates and DNA. Minor DNA damage can result in mutations that can be part of the causal chain for malignant transformation, while sustained DNA damage can result in further perturbations of cell cycle control [76]. In addition to an extensive literature on the carcinogenicity of tobacco smoke in cell and animal models, numerous case– control and cohort studies affirm its key role in man, and the super-multiplicative synergism with alcohol drinking [77]

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Alcohol Unsafe consumption of alcohol, including so-called binge drinking, is a major public health problem worldwide, for example, contributing between 5,000 and 40,000 deaths in the UK annually [78]. The possible beneficial effects of moderate alcohol consumption have been widely canvassed, because of the so-called J-shaped relationship between alcohol intake and all-cause mortality, as shown in a number of meta-analyses [79]. The upstroke of this J-curve is thought to be due to the cardio protective effect of moderate alcohol consumption: In particular, alcohol increases high density lipoprotein levels, inhibits platelet aggregation, and promotes fibrinolysis [80]. It has always been recognised that above an intake of around 10  g of alcohol per day the detrimental effects of alcohol predominate [79]. The recent increases in oral cancer reported in younger subjects in the UK were related, at least in part, to growing alcohol use/abuse in that society [44]. The difficulty of accurately quantifying the influence of alcohol in the aetiology of H&N cancer stems from the fact that most people who drink heavily also smoke. It is also difficult to obtain reliable information from individuals on their intake of alcohol. The health education council in the UK recommends a weekly intake of no more than 14 units for women and 21 units for men. Using these criteria one in four men and one in ten women in that country are believed to be drinking over this limit, with the number of habitual heavy drinkers ­estimated at four million [81]. Although the legal age for drinking is 18 years, the average age at which drinking starts has fallen since the early 1970s from around 17 to around 11  years, in boys and girls. The recent emergence of “Alcopops” (alcoholic drinks that mimic the taste of nonalcoholic drinks) has resulted in wide uptake among those under 18. Internationally there is a developing view that any consumption of alcohol is detrimental, and even the French government now publicly recommends severe constraint or even abstinence: the French National Cancer Institute has declared “there is no amount of alcohol, however small, which is good for you”[82]. WHO policy is to minimise the use of alcohol throughout all of society [83], and the 2009 Australian Guidelines to Reduce Health Risks from Drinking Alcohol summarises the science cogently [84]. Ethanol and water are the main components of most ­alcoholic beverages, which also contain volatile and nonvolatile flavour compounds. The major alcohol metabolising enzymes are alcohol dehydrogenase that oxidises ethanol to ­acetaldehyde, and aldehyde dehydrogenase that detoxifies acetaldehyde to acetate. Acetaldehyde is responsible for the oral carcinogenic effect of ethanol, owing to its multiple mutagenic effects on DNA. Specific alcoholic beverages have been shown to contain specific impurities or contaminants

N.W. Johnson and H.K. Amarasinghe

that can be carcinogenic. N-nitrosodiethylamine is present in some beer and whisky and has been associated with an increased risk of oral cancer. Polycyclic aromatic hydrocarbons, some of which are considered to be carcinogenic, are found in many brands of whisky [85]. Alcohol also acts in the following ways to promote oncogenesis [85]. Ethanol: • Damages the phospholipids of cell membranes and increases permeability. It has been shown to enhance the penetration of tobacco-specific carcinogens across the oral mucosa [86]. • Impairs DNA repair mechanisms. • Acts as a solvent, allowing the carcinogens from tobacco to penetrate into tissue. • Perhaps catalyses the activation of tobacco carcinogens. • Alcohol is highly calorific. It lessens the protective effect of beneficial foods such as fruits and vegetables by depressing hunger. • Is hepatotoxic, thus reducing the effectiveness of those enzyme systems central to detoxification of carcinogens, especially the gluthathione-S-transferases and cytochrome-p450 systems. A case–control study in Uruguay conducted between 1992 and 1996 is worthy of note [87]. Histologically confirmed cases (n = 471) of squamous cell carcinoma of the oral cavity and pharynx in males admitted to four major hospitals in Montevideo were matched with the same number of other patients admitted for a variety of non-smoking and nondrinking-related conditions as controls. Alcohol consumption was assessed by interview and the number of grams of ethanol consumed per day was calculated. Ever-drinking was associated with a 4.5-fold increased risk of oral–pharyngeal cancer compared to non-drinkers, though no clear doseresponse relationship was observed. Consumption of hard liquor was associated with a 3.6-fold increased risk, whereas pure wine drinking showed only a 2.1-fold increased risk. When risks were analysed by sub-sites, the highest odds ratios were observed for oral cavity cancer. Another case–control study conducted in Italy and Switzerland between 1992 and 1997 included 749 cases of oral/pharyngeal cancer and 1,772 hospital controls. Alcohol consumption was measured by the number of drinks consumed per day, one drink corresponding to ~125 ml of wine, 330 ml of beer or 30 ml of spirits (i.e. about 12 g of ethanol). Compared to light drinkers (1–2 drinks per day), the adjusted OR for 3–4 drinks was 2.1(95% CI 1.5–2.9) and 21.1(95%CI 14.0–31.8) for more than 12 drinks per day. Wine drinkers who consumed more than 12 drinks per day were at a 16.1fold risk compared to the abstainers. Consumption of more than 3 beers per day resulted in a 2.3-fold risk compared to the non-beer drinkers. In contrast to the Uruguayan study,

1  Epidemiology and Aetiology of Head and Neck Cancers

there was only a 1.9-fold risk for consumption of spirits as compared to non-spirit drinkers [88]. There are many confounders in such studies. Most people drink a variety of beverages, and accurate controlling for tobacco, diet, socio-economic status and other variables is challenging.

27

e­ ducation messages in most countries. The ­micronutrients that confer these benefits are also well understood. Vitamin A and related carotenoids (in particular beta-carotene), vitamins C and E and selenium appear to be particularly protective against most epithelial cancers [105–107], and much of the effect is attributable to their antioxidant activities. Antioxidants act by reducing free radical reactions which can cause DNA mutations and changes in lipid peroxidation of cellular membranes [108]. Other protective roles of micronutrients are Mouthwashes modulation of carcinogen metabolism, maintenance of appropriate cell differentiation, inhibition of cell proliferation and There has been considerable interest in the possible risks of oncogene expression, maintenance of immune function and H&N cancer associated with use of alcohol-containing inhibition of formation of endogenous carcinogens [76]. mouthwashes recently, leading some manufacturers to use A recent meta-analysis on oral cancer, based on 15 case– “alcohol-free” as a marketing tool. Epidemiological findings control studies and one cohort study, was able to utilise diet have not been consistent and control for other major risk fac- data from nearly 5,000 subjects: this estimated that each tors, including smoking, not always easy to ascertain from portion of fruit or vegetables consumed per day reduced the the published work [89]. Some reviews have argued that risk of oral cancer by around 50% [109]. These effects are using mouthwash daily may be an independent cause of can- also demonstrable with OPMD: In a population-based case– cers of the head, neck and oesophagus [90, 91]. It is well control study in Japan, where there were 48 cases of oral established that ethanol increases the permeability of lining leukoplakia and 192 control subjects, serum levels of lycomucosa, allowing carcinogens to penetrate more freely. pene and beta-carotene were significantly lower in those Acetaldehyde, the proximal metabolite of ethanol can accu- with leukoplakia; logistic regression showed that high levels mulate in the mouth from bacterial action, and as explained of beta-carotene were related to low risk of oral leukoplakia above this is an established carcinogen. However, four case– (OR = 0.16) [110]. control studies have shown non-significant, lower or similar Intervention studies are also encouraging in this respect. oral cancer risks among self-reported mouth wash users In a major double-blind placebo-controlled trial in Kerala compared to non-users [92, 93]. The most recent meta-­ [111], up to one third of subjects showed regression of their analysis has not demonstrated excess risk for oral cancer oral leukoplakias after 12 months supplementation with oral from alcohol-containing mouthwashes [94, 95]. There is, beta-carotene. Extensive studies from the MD Anderson however, a plausible biological basis for risk associated with Cancer Centre in the USA are progressively identifying the alcohol-containing mouthwashes, especially in smokers and most effective combinations of anti-oxidants in the regresit is always prudent to remember that absence of evidence is sion of OPMD and the prevention of recurrences and second not evidence for absence. primary neoplasms in H&N cancer, although it has to be recognised that these agents do not always prevent the progression of an OPMD to overt cancer [112]. There is current interest in the protective effects of tea, Diet and Nutrition in the Aetiology especially green tea, which contains high levels of polypheof Head and Neck Cancer nols [113]. These are powerful antioxidants able to ­counteract both initiation and promotion of carcinogenesis [108]. Dietary factors are estimated to account for approximately 30% of all cancers in Western countries [96]. This proportion is currently thought to be about 20% in developing countries and is projected to increase in the future [97]. Poor diet is a Genetic Predisposition significant risk factor for all H&N cancers [98–104] and appears to be second only to tobacco as a cause of oral can- There is considerable evidence for a minor component of cers worldwide [3]. A case–control study of laryngeal cancer inherited, genetic predisposition in UADT cancers, related to in Italy and Switzerland between 1992 and 2000, revealed polymorphisms in carcinogen-metabolising enzyme systems that a diet not only rich in, but also varied in, fruit and vege- [114]. A recent extensive meta-analysis [91] pooled individtables confers decreased risk of laryngeal cancer [101]. ual-level data across 12 case–control studies including 8,967 Evidence comes from case–control and cohort studies, from HNC cases and 13,627 controls. After adjusting for potential animal and from in vitro experiments. Protective and unhealthy confounding factors a family history of H&N cancer in firstfoods are well understood, and form the basis of health degree relatives increased the risk (OR = 1.7, 95% CI 1.2–2.3).

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N.W. Johnson and H.K. Amarasinghe

The risk was higher when the affected relative was a sibling (OR = 2.2, 95% CI 1.6–3.1) rather than a parent (OR = 1.5, 95% CI 1.1–1.8) and for more distal H & N sites (hypopharynx and larynx). The OR rose to 7.2 (95% CI 5.5–9.5) among subjects with family history, who were alcohol and tobacco users. No association was observed for family history of nontobacco-related neoplasms and the risk of HNC (OR = 1.0, 95% CI 0.9–1.1). Rare cancer syndromes can involve the H&N: Cowden syndrome, caused by mutations in the tumour suppressor gene PTEN; and dyskeratosis congenita, in which oral white lesions in young people have a risk of malignant transformation [115].

and heating is a significant health problem in many ­developing countries, where a greater proportion of people use such fuels frequently in poorly ventilated areas. Many studies have been identified indoor air pollution as a risk factor for H&N cancer [123, 124] and a recent monograph by the International Agency for Research on Cancer has identified indoor air pollution from coal usage as a known human carcinogen, while that from biomass (primarily wood) as a probable human carcinogen [125]. Studies carried out in China and Brazil have reported exposure to wood smoke as a risk factor for oral cancer [126], nasopharyngeal cancer [127] and UADT cancer [128].

Microorganisms

Solar Radiation

Microorganisms have been implicated in the aetiology of oral leukoplakia for more than a century, beginning with the classic dorsal leukoplakia of syphilitic glossitis. Today tertiary syphilis is rare, but the fungus, Candida albicans, a common oral commensal, is frequently found invading the upper epithelium in histological sections of leukoplakia, more so in the mouth than pharynx or larynx [116], and this involvement is associated with a higher risk of malignant transformation [117]. The terms “candidal leukoplakia” and “hyperplastic candidiasis” have been used to describe such lesions. It is now clear that high-risk HPV genotypes, particularly HPV 16 and 18, are important co-factors, especially in cancers of the tonsil and elsewhere in the oropharynx [118, 119]. The current state of knowledge is covered extensively in another chapter of the present volume. The role of bacteria in the aetiology of UADT cancers is currently receiving more attention [120]. Endogenous production of acetaldehyde and reduction of nitrate to nitrites by oral flora is higher in drinkers with poor oral hygiene [121]. Understanding the role of the oral flora is certainly important in the management of the distressing mucositis associated with so much cancer therapy.

Prolonged exposure to sunlight represents an important risk factor for the development of squamous cell carcinoma of the lip in people with fair complexions, and those with outdoor occupations. Usually, the lower lip is involved because it receives considerably more direct sunlight than the upper lip [129]. Evidence comes from many countries, including those at latitudes with clean air through which ultraviolet light penetrates easily, such as Finland [130] or Sweden [131], and from countries closer to the equator with regular long hours of sunshine such as rural Greece where lip cancer can account for 60% of oral cancers [132] and in India, for example, in fishermen [6] – though some protection may exist in darker-skinned races or individuals. In Finland, the increased risk for lip cancer is confounded by smoking and social class, whereas that for oral cavity and pharynx is not; at these latter sites, alcohol was a much stronger confounder than tobacco [133]. A study from California shows that risk for women is strongly related to lifetime solar radiation exposure, but lipstick and other sunscreens are protective [134]. Although the observation goes back over a decade, there is a recent concern that modern cosmetic lip glosses may enhance UV damage to the lips, including increased risk of cancer [134]. Falls in the incidence of lip cancer have been interpreted as due to reduced occupational exposure to sunlight and to reduced pipe and cigar smoking [135, 136].

Air Pollution Part of the urban/rural difference in the incidence of head and neck cancer has been related to atmospheric pollution. For example, mean sulphur dioxide and smoke concentrations in the atmosphere are positively correlated with squamous cancer of the larynx and, to a lesser extent, the pharynx in data collected some time ago from the West Midland region of England 1950–1990 [122]. Indoor air pollution resulting from the use of solid fuel such as wood, crop residue, animal dung and coal for ­cooking

Global Scenario of Oral Potentially Malignant Disorders and Laryngeal Leukoplakia The term Oral Potentially Malignant Disorders (OPMD) was recommended by an international Working Group convened by the WHO Collaborating Centre for Oral Cancer and Precancer in London in 2005 [135]. It conveys that not all

1  Epidemiology and Aetiology of Head and Neck Cancers

29

disorders described under this umbrella will transform to invasive cancer – at least not within the lifespan of the affected individual. Leukoplakia, Erythroplakia, Oral Submucous fibrosis, Lichen planus, Palatal lesions in reverse smokers, Actinic keratosis, Discoid lupus erythematosus, Dyskeratosis congenita, and Epidermolysis bullosa are described under the broad definition of OPMD [137, 138].

Global Prevalence of OPMD Estimates of the global prevalence of OPMD range from 1 to 5% [139], although much higher prevalences are reported from Southeast Asia, usually with a male preponderance, for example, in Sri Lanka (11.3%) [50], Taiwan (12.7%) [140]

and Pacific countries like Papua New Guinea (11.7%) [141]. Wide geographical variations across countries and regions are mainly due to differences in socio-demographic characteristics, the type and pattern of tobacco use and clinical definitions of disease (see Table 1.4). In Western countries, the overall prevalence is low and a decreasing trend over time is observed. Stefano [154], conducted a meta-analysis of 23 primary studies on oral leukoplakia, from international data published between 1986 and 2002. The point-prevalence estimates were 1.49% (95% CI 1.42–1.56%) and 2.6% (random effect, 95% CI 1.72–2.74%). Leukoplakia was significantly more prevalent among males (prevalence ratio 3.22), but no difference was found between geographical areas and between younger and older adults. Using these data, they calculated that the crude annual oral cancer incidence rate attributable

Table 1.4  Summary of the prevalence of OPMD reported in the literature References Country (year) Sampling method F/M ratio Age group

Disease entity

Definition used

Prevalence %

³30

OPMD

WHO 1994

0.9/1.0

³15

Not given

0.9/1.0

³20

OPMD Leukoplakia Erythroplakia Lichen planus OSF Leukoplakia

11.3 weighted for gender and geographical location. 12.7 7.4 1.9 2.9 1.6 0.5–0.3

[50]

Sri Lanka (2008) MSSC

0.6/1.0

[142]

Taiwan (2005)

Random

[143]

USA (2003)

MSSC

[144]

Sri Lanka (2003) Multi-stage stratified – cluster (MSSC)

[145]

Spain (2002)

Stratified, random

0.8/1.0

35–44 years and OPMD 65–74 years Leukoplakia Erythoplakia OSF ³30 Leukoplakia

[146]

Germany (2000)

Stratified, random

1.0/1.0 0.7/1.0

35–44 year 65–74 year

Leukoplakia Leukoplakia

[147]

Japan (2000)

All invited

0.4/1.0

m >40, f >20

[148]

Malaysia (1997)

Stratified, random

0.7/1.0

³25

[149]

Netherland (1996)

Waiting room

0.9/1.0

13–93 year

Leukoplakia Lichen planus Leukoplakia Erythroplakia OSF Lichen planus Leukoplakia

[150]

Hungary (1991)

Random

0.7/1.0

All age groups

[151] [152] [153]

Japan (1991) Sweden (1987) Sweden (1987)

Factory workers Stratified random All-invited residents

0.5/1.0 18–63 years Not found ³15 0.9/1.0 ³15

Leukoplakia Lichen planus Leukoplakia Lichen planus Leukoplakia

Kramer 1978, Kramer 1980 WHO 1994

WHO 1978, Axell, T et al. 1984 Axell 1976 Zain 1995 WHO-ICD-DA WHO 1980 WHO 1978 Axell, T et al. 1984

Axell 1984 Axell 1996 Schepman 1995 Axell 1984 Axell 1984 Axell 1976 Axell 1976

4.1 2.6 0.4 1.6 1.6 1.0 0.19 0.21 0.96 0.01 0.06 0.38 0.6

1.3 0.1 2.5 1.9 3.6

30

to leukoplakia would be between 6.2 and 29.1 per 100,000 thus suggesting that the global number of oral cancer cases is probably under-reported.

Age and Gender Distribution of OPMD This varies considerably, mainly dependent on lifestyle and thus on ethnicity and geographical location. In the developed world, leukoplakia is usually found between the fourth and seventh decades of life, in the developing world some 5–10 years earlier [155]. Females are less commonly affected, largely reflecting greater use of relevant habits by men.

Malignant Transformation of OPMD Risk of malignant transformation varies from site to site within the mouth, from population to population and from study to study [156–158]. A classic study conducted in the 1970s with follow-up over 7  years of more than 30,000 Indian villagers, showed transformation rates from 10 to 24 per 100,000 per year [157]. Another classic study from the early 1980s, a hospital-based study in Californian patients with oral leukoplakia, with a mean follow-up period 7.2 years, revealed a malignant transformation rate of 17.5% [158]. Rates for hospital-based studies are, unsurprisingly, consistently higher than community-based studies because of sampling bias. Petti [154] has estimated a mean global prevalence of 2.6% for leukoplakia, and a mean global transformation rate of 1.36% per year (95% CI 0.69–2.03). Extrapolating from these figures suggests that considerably more OSCC should have been reported in recent times, a possible reason being underreporting of cases of oral cancer in the developing world.

Epidemiology of Laryngeal Leukoplakia Epithelial precursor lesions of the larynx, clinically defined as leukoplakia and chronic laryngitis, are mostly seen in adults and affect men more often than women. This gender disparity is more pronounced after the sixth decade of life [159]. Epidemiological studies of laryngeal precursor lesions are scarce and the incidence differs worldwide and depends upon the amount, manner and types of exposure to relevant carcinogens. According to a recent review [160], 1,268 patients were clinically diagnosed as laryngeal leukoplakia and chronic laryngitis during the period from 1979 to 2004 in Slovenia. The incidence of patients, covering a region

N.W. Johnson and H.K. Amarasinghe

with approximately 800,000 inhabitants or 40% of the population of Slovenia, varied for the benign group of precursor lesions (squamous hyperplasia and basal parabasal call hyperplasia) from 0.84 to 4.62/100,000 inhabitants pa (mean value 2.61/100,000 inhabitants, SD = 1.10). The incidence of patients for atypical hyperplasia ranged from 0.25 to 2.62/100,000 inhabitants pa (mean value 0.86/100,000 inhabitants, SD = 0.49).

Aetiology of Laryngeal Leukoplakia Laryngeal leukoplakic lesions are strongly associated with tobacco smoking and alcohol use, especially in combination [161–163]. Other risk factors are: industrial pollution, specific occupational exposures, nutritional deficiency, and hormonal disturbance [164–166]. A recent meta-analysis has shown a weak association between HPV-16 and laryngeal cancer [167]. Several authors have recently devoted much attention to the potential role of gastro-esophageal reflux disease, but the results are not conclusive [161, 168].

Salivary Gland Neoplasms Epidemiology Neoplasms arising in the salivary glands are relatively uncommon, yet they represent a wide variety of both benign and malignant histologic sub-types. The reported annual incidence, when all salivary gland tumours are considered, varies widely between countries and regions [169]. According to Globocan 2002, the world’s highest incidence of salivary neoplasms was reported from the Northern Territory of Australia (though the number of cases in this thinly populated area was too small – only seven cases – to place credence on this value); the second highest from Croatia (Table  1.5). Within Japan, the highest rates are reported from the region of Nagasaki, regarded as long-term effects of the atomic bomb explosion in 1945. The estimated annual incidence in the USA is 1.5 cases per 100,000 population pa; here they constitute only about 6% of all head and neck neoplasms [170].

Site, Age and Sex Distribution Nearly 80% of these tumours arise in parotid glands, 15% in submandibular glands, with the remainder distributed across the sublingual and minor salivary glands of the oral and

1  Epidemiology and Aetiology of Head and Neck Cancers

oropharyngeal mucosae [171]. In most series, benign ­neoplasms are the majority, representing 54–79% of cases described. Pleomorphic adenoma is by far the most common, accounting for about 50% of all salivary gland tumours. Warthin’s tumour is second in frequency among benign neoplasms and, in most large studies, mucoepidermoid carcinoma is the most common malignancy [169].

Table  1.5  Incidence of salivary neoplasms: cases per 100,000 pa, standardised ASR(W) Population ASRW male ASRW female Australia, Northern territory Croatia Poland, Cracow USA, District of Colombia white USA, black USA, white UK, Oxford region Canada China, Hong Kong India, Chennai Japan, Nagasaki France, Herault Norway Spain, Granada Switzerland, Geneva

1.7 1.6 1.5 1.4 0.9 1.1 0.7 0.9 0.7 0.5 0.7 0.8 0.7 0.6 0.6

0.2 0.6 0.6 0.4 0.6 0.8 0.5 0.6 0.5 0.3 0.5 0.7 0.6 0.5 0.9

Fig.  1.26  The incidence of salivary gland neoplasms rises steadily, and linearly, with age. Data from selected countries are given in Fig. 1.26 (a, males; b, females). Note that the scales are, as usual with

31

The average ages of patients with benign or malignant tumours are 46 and 47  years, respectively, with peak incidence of most of the specific types in the sixth and seventh decades. However, the highest incidence of pleomorphic adenomas, mucoepidermoid carcinomas and acinic cell carcinomas is significantly younger in the third and fourth decades. Salivary neoplasms are rare in young people and in patients under 17  years of age, a neoplasm of a major gland is as likely to be mesenchymal as epithelial in origin [172–175] (Figs. 1.26a, b).

Aetiology of Salivary Gland Neoplasms The aetiology of salivary gland neoplasms is still poorly understood. Furthermore, especially with neoplasms that have mixed cellularity, notably pleomorphic adenomas and carcinomas arising therein, which show epithelial, myoepithelial and mesenchymal characteristics, controversy remains as to whether there is a single or more than one type of cancer stem cell [169]. Viruses: Studies have shown a strong association between EBV and lymphoepithelial carcinomas [176, 177], with geographical variations, as this shows a preponderance for Asian

such data presentations, logarithmic. Thus it is seen that across most of the life span there is no major sex predilection

32

patients [178] and Greenlandic Inuits [179]. Salivary tissue is an established reservoir for EBV, but a clear oncogenic role for EBV or for cytomegalovirus (CMV) has not being demonstrated in other salivary gland carcinomas or in benign parotid tumours [177]. SV40 sequences have been postulated in human pleomorphic adenomas [180], but there is no significant association between human salivary gland tumours and other viruses, including polyoma virus and papillomavirus (HPV).

N.W. Johnson and H.K. Amarasinghe

some studies [205], but were absent in another [206]. Progesterone and androgen receptors are present in some salivary neoplasms [205, 207] and binding of hormones to these may influence tumour progression.

Other Important Cancers of the Head and Neck: Malignant Melanoma Radiation: There is convincing evidence implicating expo- and Kaposi’s Sarcoma (KS) sure to ionising radiation and the development of salivary gland neoplasms. Long-term follow-up studies of the survivors of the atomic bomb explosions in Hiroshima and Nagasaki show an increased relative risk of 3.5 for benign, and 11 for malignant salivary neoplasms [181, 182]. The risk was directly related to the level of exposure to ionising radiation. There was a high frequency of both mucoepidermoid carcinoma and Warthin’s tumours in these patients [183]. Therapeutic radiation, especially in the head and neck region, has been linked to significantly increased risk [184, 185]. Iodine 131, used in the treatment of thyroid disease, is thought to produce neoplasms, as the isotope is also concentrated in salivary glands [186]. Several studies have suggested that exposure to routine dental radiographs may be associated with an increased risk of salivary neoplasms, though the evidence is inconclusive [187, 188]. Exposure to ultraviolet radiation has also been implicated [189–191], though this seems biologically improbable. There appears to be no excess risk in those exposed to radon [192], or the microwaves of cellular telephones [193, 194]. Occupation: There is a literature relating salivary gland neoplasms to occupation. Suggested risks include rubber manufacturing [195], exposure to metal in the plumbing industry [196] and nickel compounds [195], woodworking in the automobile industry [197] and employment in hairdressing and beauty shops [198, 199]. An increased risk of salivary gland cancers was reported in people living in certain Quebec countries where asbestos was mined, and this risk was inversely proportional to distance from the mines [200]. Lifestyle and nutrition: Tobacco and alcohol, which are highly associated with head and neck squamous cell carcinoma, have not been shown to play a major role in the development of salivary malignancies [201]. However, tobacco smoking has been associated with the development of Warthin’s tumour. Exposure to silica dust and kerosene as a cooking fluid increased the risk of salivary neoplasms in a Chinese population [202], and an increased risk of parotid neoplasms was associated with exposure to nickel, chromium, asbestos and cement dust [203]. An elevated level of risk has been described in those with a high cholesterol intake [204]. Hormones: Oestrogen activity or upregulation of oestrogen receptors have been described in pleomorphic adenomas in

Malignant melanoma is recorded by cancer registries separately from mucosal and other cancers (Table  1.6). These data represent all skin sites but the management of melanoma often falls into the hands of head and neck clinicians, so the data are of interest. DNA damage from ultraviolet light, especially acute sunburn and especially early in life, is the major risk factor. This explains the high incidence rates in Australia, New Zealand, northern Europe and among white South Africans: for head and neck melanoma the risks associated with ultraviolet light are most marked at low altitudes [208]. Melanoma of UADT mucosa is a serious, usually fatal, disease: global epidemiological data will be “buried” in the graphs and tables above. Such data as available have been reviewed recently by van der Waal et al. [209].

Table 1.6  World standardised incidence rate per 100,000. Accessed from http://www-dep.iarc.fr/ in December 2009 Melanoma skin (C43) Kaposi sarcoma (C46) Country Male Female Male Female World More developed Less developed Eastern Africa Middle Africa Northern Africa Southern Africa Western Africa Caribbean Central America South America Northern America Eastern Asia Southeastern Asia South Central Asia Western Asia Eastern Europe Northern Europe Southern Europe Western Europe Australia New Zealand Melanesia Micronesia Polynesia

2.8 8.3 0.7 1.2 2.2 0.7 5.4 1.1 1.0 1.3 2.4 16.4 0.3 0.5 0.5 1.6 3.3 8.4 6.0 7.3 38.5 33.8 4.8 1.2 5.1

2.6 7.5 0.7 2.3 2.1 0.5 4.1 0.9 1.1 1.7 2.3 11.7 0.2 0.5 0.4 1.5 3.8 10.0 5.5 10.3 29.5 29.2 2.9 0.7 0.0

0.0 0.0 0.0 23.0 30.0 0.3 13.2 4.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 9.5 8.6 0.1 5.7 1.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

1  Epidemiology and Aetiology of Head and Neck Cancers

Kaposi’s sarcoma (all sites) is an AIDS-defining lesion and is thus most common where HIV-disease is most rampant: it is a major problem in sub-Saharan Africa, in many countries of which KS is the most frequently diagnosed cancer [210]. In our series of 710 head and neck cancers in northern Nigeria, KS was the most common HIV-associated malignancy [211]. KS is seen less commonly in the current era of highly active anti-retroviral therapy in populations where such therapy is widely available. Many of the zero numbers in these tables reflect absence of data – or situations where KS is not separately registered. The aetiology of KS was described in 1994 and is now clearly established as infection with Human Herpes Virus Type 8 (HHV-8, also known as Kaposi Sarcoma Herpes Virus – KSHV). It is a multifocal malignancy of lymphatic endothelial cells. Endemic KS in HIV-negative subjects still exists, especially in the Mediterranean where it has long been regarded as having an ethnic predilection – for certain Jewish groups. There is a puzzle with HIV/AIDS-related KS, however; the head and neck, especially the mouth, is a common site for KS in HIV-positive subjects; the oropharynx is the primary reservoir, and saliva/oral fluids are the major vehicle of transmission [212]. Transmission occurs via oral-genital contact and is more common in men who have sex with men. In India, which is currently the single nation of the world with the highest number of HIV infections, KS is almost never seen. Whether this is because of different social practices, differences in the strains of KS circulating in that country – with different pathogenicity – or differences in host response, remains unknown [213] (Table 1.7). The Death to Registration Ratio (D/R) for melanoma can be readily calculated here. For ANZ this ranges from 0.09 to 0.18, whereas in northern Europe, the average approaches double this, viz 0.16 for women and 0.26 for men. Women do better all over the world, possibly because they seek treatment earlier. Note that these outcomes are substantially ­better than for oral cancer. In ANZ, there are highly effective public education campaigns regarding protection against sun ­damage, and many screening and treatment facilities. In spite of this, the comparatively poor outcomes perhaps reflect a degree of complacency towards the very common suninduced lesions, many of which are benign.

Primary Neoplasms of the Jaws and Facial Bones While to a large extent, these lesions constitute the “bread and butter” for many oral/maxillofacial pathologists and ­surgeons, such lesions are comparatively rare: they do not represent anything like the major public health problem of epithelial tumours of the head and neck. They are not, ­therefore,

33 Table  1.7  World standardised mortality rate per 100,000. Accessed from http://www-dep.iarc.fr/ in December 2009 Melanoma skin (C43) Kaposi sarcoma (C46) Country Male Female Male Female World More developed Less developed Eastern Africa Middle Africa Northern Africa Southern Africa Western Africa Caribbean Central America South America Northern America Eastern Asia Southeastern Asia South Central Asia Western Asia Eastern Europe Northern Europe Southern Europe Western Europe Australia New Zealand Melanesia Micronesia Polynesia

0.8 1.8 0.3 0.7 1.3 0.4 2.9 0.7 0.4 0.6 0.9 2.5 0.1 0.3 0.3 0.7 1.6 2.2 1.6 1.8 5.1 6.1 2.5 0.6 2.5

0.6 1.2 0.3 1.3 1.3 0.3 2.2 0.5 0.3 0.5 0.6 1.3 0.1 0.3 0.2 0.6 1.2 1.6 1.1 1.3 2.6 3.6 1.5 0.3 0.0

0.0 0.0 0.0 20.8 25.3 0.2 12.4 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 8.8 7.8 0.1 5.4 1.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

a major thrust of this volume, but have excellent coverage in other modern textbooks including those referred to in the “Introduction” of this chapter. It is not appropriate here to indulge in the favourite pastime of oral pathologists to debate the classification of such lesions, uniformity of which would be essential to the comparability of international epidemiological data. Furthermore, it is extremely difficult to mine international and national databases for detailed histological typing, so that the incidence and mortality associated with bone and odontogenic tumours might be reliably quantified. Recourse has to be made to case series and, while these are valuable, significant regional differences in epidemiology and risk factors are hard to quantify. A concise summary of the situation with odontogenic tumours is in the WHO “Blue Book” of 2005 [169]. Difficulties also arise because some databases/case series include benign neoplasms – and with odontogenic lesions, there are frequently grey areas regarding the behaviour of a particular diagnostic category or individual lesion. Strictly speaking, cancer registries should only record malignancy. Hamartomatous and benign lesions are very much more common than malignant odontogenic tumours [214]: Differences emerge between case series based on dental/oral-maxillofacial departments which are more likely to include the former, whereas cases handled in broader general hospitals or cancer hospitals will select for malignancies.

34

Ameloblastoma is clearly the most common malignant odontogenic tumour worldwide. An extensive series of 1,642 cases from Sichuan University [215] found that benign tumours comprised 97% of cases: ameloblastoma was the most common malignancy, followed by odontogenic keratocystic tumour. In a series of 1,088 cases from northern California [216], 76% were (benign) odontomas: ameloblastomas comprised 12% – a surprisingly high figure perhaps reflecting the specialised nature of this laboratory. This paper also tabulates data from case series all over the world describing the frequencies of the various types of odontogenic “tumour”. There has long been an impression that odontogenic tumours are more common in Africa – perhaps because so many advanced lesions come late to diagnosis. A thoughtful analysis of the literature up to the early 1990s is given by Smith, [217]. In a more recent series, of 308 odontogenic tumours in Lagos, southern Nigeria, 97% of the tumours were benign and only 3.4% malignant; ameloblastoma with predilection for the mandible was the most frequent [218]. Among primary malignant bone tumours [219], most case series around the world contain very small numbers of patients, but indicate various types of osteo[genic] sarcoma to be most common. Osteosarcomas of all sites account for 40–60% of primary malignant bone tumours and ~10% of these occur in the head and neck, mostly in the jaws. These tend to be diagnosed approximately two decades later than their long bone counterparts, which have a peak incidence between 10 and 14  years. Head and neck osteosarcomas metastasise less frequently than those in long bones, and have a better 5-year survival rate, reported between 27 and 84%. The experience of one USA centre has recently been described [220], with a helpful review of the literature. Out of 2,830 biopsies of oral and jaw lesions diagnosed 1983– 2003, in Lagos, 59 (2.08%) were primary malignant bone tumours, osteosarcoma again being most frequent (28.8%). Interestingly the mean age at presentation (27 ± 14  years) was lower than reports from other parts of the world.

N.W. Johnson and H.K. Amarasinghe

followed by 22% on the tongue: The role of ­inflammation in the attraction of metastatic cells to the gingiva has been suggested [222].

The Future of Head and Cancer Epidemiology As with many aspects of life, global inequalities are increasing in the incidence rates of head and neck cancers, in the provision and quality of prevention and screening programmes, and in access to and quality of patient care. The drivers of these inequalities are socio-political: war, poverty, pestilence, climate change, lack of food and water security [223]. The problems do not derive primarily from ignorance of causes and mechanisms of disease, but from ineffective or absent implementation of the right policies, and from lack of resources to implement them. As scientists and clinicians devoted to head and neck oncology we all have a moral responsibility to contribute to these wider social and political challenges. The knowledge to apply world best practice is within the pages of this book. The leadership of many local and national bodies is acknowledged: these activities need to be in dialogue and synergy with global leadership through agencies such as the World Health Organisation, the International Agency for Research on Cancer, the UICC/International Union against Cancer, the International Federation of Head and Neck Oncologic Societies, the International Academy of Oral Oncology and others. The International Association for Dental Research launched an initiative in 2010 seeking to reduce global inequalities in oral cancer (and in other oro-facial diseases and disorders). It is a truism that however sophisticated and effective our diagnostic and treatment armamentarium becomes, head and neck cancer rates around the world will never be reduced by such interventions – though, of course, hundreds of thousands of lives may be saved or improved. The emphasis must be on primary and secondary prevention, on the implementation of policies that work to these ends and on their continued evaluation and improvement.

Cancer Metastatic to the Head and Neck Tumours metastatic to the H&N from distant sites are comparatively rare, representing about 1% of oral tumours. Most lesions are found in patients between the fifth and seventh decades of life. They affect the jaws more commonly than soft tissues in a ratio of 2:1 [221]. The most common primary tumours metastatic to the jaws are breast (20%), lung (13%), kidney (8%), adrenal (8%), bone (7%), colorectal (6%), prostate (5%), and liver (5%). A review of cases revealed that 54% of the 218 metastatic tumours to oral soft tissues were located on the attached ­gingiva,

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40 217. Smith CJ. Odontogenic Neoplasms and Hamartomas. In: Prabhu R, Wilson DF, Daftary DK, Johnson NW, editors. Chapter 33 in: Oral Diseases in the Tropics. New York: Oxford University Press; 1992. 218. Ladeinde AL, Ajayi OF, Ogunlewe MO, Adeyemo WL, Arotiba GT, Bamgbose BO, et al. Odontogenic tumors: a review of 319 cases in a Nigerian teaching hospital. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2005;99:191–5. 219. Ogunlewe MO, Ajayi OF, Adeyemo WL, Ladeinde AL, James O. Osteogenic sarcoma of the jaw bones: a single institution experience over a 21-year period. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101:76–81.

N.W. Johnson and H.K. Amarasinghe 220. Fernandes R, Nikitakis NG, Pazoki A, Ord RA. Osteogenic ­sarcoma of the jaw: a 10-year experience. J Oral Maxillofac Surg. 2007;65:1286–91. 221. Hirshberg A, Shnaiderman-Shapiro A, Kaplan I, Berger R. Metastatic tumours to the oral cavity – pathogenesis and analysis of 673 cases. Oral Oncol. 2008;44:743–52. 222. Hirshberg A, Buchner A. Metastatic tumours to the oral region. An overview. Eur J Cancer B Oral Oncol. 1995;31B:355–60. 223. Hobdell MH, Oliveira ER, Bautista R, Myburgh NG, Lalloo R, Narendran S, et al. Oral diseases and socio-economic status (SES). Br Dent J. 2003;194:91–6. discussion 88.

Chapter 2

Head and Neck Cancer Prevention Fausto Chiesa, Angelo Ostuni, Roberto Grigolato, and Luca Calabrese

Abstract  Head and neck cancer (HNC) represents a broad spectrum of diseases that involves the nasal and oropharyngeal cavities, the paranasal sinuses, the major and minor salivary glands, the larynx and the lymphatic tissues of the neck. The world-wide yearly incidence exceeds over half a million cases. Tobacco (smoking and smokeless) and alcohol use are the principal risk factors, however, a substantial and increasing proportion of head and neck tumors cannot be attributed to these. Recent evidence has shown that the incidence of oropharyngeal cancer among women and younger patients continues to grow and it is not related to alcohol or tobacco use but to human papillomavirus infection. Substantial advances in treatment regimens made over the last two decades have not improved the 5-year mortality rate that remains approximately 50%. Prevention represents the best opportunity to improve oncologic results and it consists of three levels of intervention: primary prevention (considered the best) aims to avoid exposure to established risk factors; secondary prevention consists of early diagnosis; tertiary prevention involves active management of patients already treated for HNC. In this chapter, we review the natural history of oral cavity and laryngeal cancer as well as the known mechanisms of carcinogenesis. Precancer and risk markers for cancer are discussed as they relate to prevention in all its forms (primary, secondary, and tertiary). Chemoprevention is the use of natural or synthetic chemicals to reverse, suppress, or prevent the conversion of a premalignant lesion to a true neoplasm. It spans all three forms of prevention and it can aim at both local and locoregional disease control. All of the major important chemoprevention clinical trials reported on in the scientific literature are presented and discussed critically and their impact on clinical practice is presented.

F. Chiesa (*) Department of Head and Neck Surgery, European Institute of Oncology, Via Ripamonti, 435, Milano 20141, Italy e-mail: [email protected]

Attention is given to new directions in the field and how HNC prevention may progress through the search for new, sensitive, and specific biomarkers as well as an improved understanding of the biomolecular mechanisms of tumor invasion, metastasis, and the newly acquired data from the Human Genome Project. Improvement in HNC prevention requires a multidisciplinary approach to face complex processes and multiple factors that may act concurrently in the etiology of disease. Future challenges remain in the correct interpretation of new findings and their wise and scientific application. Only then will we be able to impact the field of HNC, transforming prevention in the only form of cure. Keywords  Prevention • Early diagnosis • Chemoprevention • Precancerous lesions • Risk factors • HPV • Biomarkers • Molecular medicine • Multidisciplinary approach

Introduction Head and neck cancer (HNC) represents a broad spectrum of diseases that involves the nasal and oropharyngeal cavities, the paranasal sinuses, the major and minor salivary glands, the larynx and the lymphatic tissues of the neck. The worldwide yearly incidence exceeds over half a million cases [1]. Tobacco (smoking and smokeless) and alcohol use are the principal risk factors, however, a substantial and increasing proportion of head and neck tumors cannot be attributed to these. Recent evidence has shown that the incidence of oropharyngeal cancer among women and younger patients continues to grow and it is not related to alcohol or tobacco use but to human papillomavirus (HPV) infection [1–5]. Substantial advances in treatment regimens made over the last two decades have not changed the 5-year mortality rate that remains approximately 50% [6–11]. The diagnosis of HNC is often dramatically delayed in spite of easy access for evaluation and screening [12–14]. Late diagnosis results in complex, aggressive, and often mutilating treatment with a

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_2, © Springer Science+Business Media, LLC 2011

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high morbidity and significant functional compromise. Local disease control (e.g., minimizing metastases and managing recurrence) and development of a second primary tumor remain two of the most significant challenges [15, 16]. In fact, second primary tumors are among the major cause of morbidity and mortality among patients cured for head and neck squamous cell carcinomas (HNSCC). Prevention of HNC could offer the best opportunity to improve oncologic results and it consists of three levels of intervention. Primary prevention aims at avoiding exposure to established risk factors. Approximately 80% of HNCs are tobacco and alcohol related [1–3]; this percentage is not so easy to reduce because of the addiction induced by their daily use and the powerful impact of advertising by the tobacco and liquor industry particularly on the younger population. The increased incidence of HPV-related cancers has been linked to a change in the sexual patterns in the overall population. Currently, other than monogamous sexual intercourse and avoidance of orogenital intercourse, no effective strategies exist to eliminate this risk factor. Secondary prevention consists of early diagnosis. Early detection programs usually entail regular clinical evaluation of asymptomatic at-risk patients; consistent and reliable instrumental or serologic tools are currently unavailable. Even though screening is not equally successful for all HNCs, the premise is that early diagnosis could improve morbidity and mortality outcomes. Improved screening increases the overall number of diagnoses, however, in order to be truly effective, it must be associated with increased disease-free survival, a decreased mortality rate, and improvement in the effectiveness of treatments. If this is not possible, and the patient’s quality of life does not improve, the cost–benefit ratio may be too high to be justified [17]. Tertiary prevention involves management of patients already treated for HNC. The interventions range from educational programs to smoking cessation for those patients who continue to smoke even with the diagnosis of a malignancy and include early diagnosis of recurrences and/or second primary tumors.

Natural History of Head and Neck Cancers Head and Neck Carcinogenesis The development of HNCs is generally related to field cancerization and multistep carcinogenesis. Field cancerization is a morphological concept arising from Slaughter’s observation that in all resected oral tumors, the macroscopically

F. Chiesa et al.

benign epithelium beyond the periphery of the primary tumor was microscopically abnormal [18]. Exposure of an epithelial field to repeated carcinogenic insults results in the development of genetic damage to normal-appearing mucosa. The entire field is susceptible to multifocal development of squamous intraepithelial neoplasia (SIN) and cancer [18–21]. A distinct but related concept is “the field of tissue injury,” which includes the molecular changes occurring throughout the tissue exposed to a carcinogen [22]. The field of injury reflects the host’s response to and damage from the carcinogen; this may or may not be a precursor to premalignant lesions and frank malignancy. Field cancerization and the field of injury have both been implicated in many malignancies and potentially hold the keys for preventing and curing epithelial cancers and for understanding in vivo epithelial carcinogenesis. Target treatments to reduce cancer risk involve the whole field. On a molecular level cancer is considered a disease of genetic, progressive, multistep mutation [23–29], however, carcinogenesis may take multiple paths and may be multifocal. This progression is heralded in tissues by the appearance of associated specific molecular and genotypic damage resulting in phenotypic changes that progress from normal histology to early dysplasia, continuing on to severe dysplasia, superficial cancers, and finally invasive disease [23, 24]. It has been estimated that four to six genetic events are required to progress from severe dysplasia to cancer and that one HNC could require up to 10–20 years to develop. The degenerative advance of cancer, however, is not always linear or sequentially additive: progression can occur away from clinically visible lesions, strongly suggesting that genetic aberrations may not always result in locally apparent disease and accumulation of mutations. Lesions that appear morphologically similar harbor often different molecular ­fingerprints, suggesting that a given phenotypic change can arise from diverse pathways. This absence of a direct, predictable, and consistent correlation between clinical and histological features of suspect lesions is well documented [23–29]. Recent microarray investigations of chromosomal aberration patterns of HPV-negative oral and oropharyngeal squamous cell carcinomas showed subclasses of cancer with unique genetic and clinical fingerprints. This observation, if confirmed in larger studies, could have important diagnostic and therapeutic implication in clinical practice [30].

Precancerous Lesions Epidemiological, experimental, and clinical observations teach us that cancer may be preceded by a morphological tissue modification, a precancerous lesion, clinically manifest

2  Head and Neck Cancer Prevention

as a white (leukoplakia), a red (erythroplakia), or a red-white lesion (erythro-leukoplakia).

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initial biopsies and the examination of definitive surgical specimens may strongly influence the decision-making process when assessing and managing suspicious lesions [42, 44].

Oral Cavity Leukoplakias and Related Lesions White lesions in the oral cavity were thought to be precancerous as early as 1870 by Paget, who described them as ichthyosis, smoker’s patch, and leucokeratosis [31]. The term leukoplakia was first used by Schwimmer in 1877 [32]. In 1936, McCarthy described the microscopic features of oral leukoplakias, grading them as 1–4, where grade 4 referred to lesions showing microscopic evidence of significant dysplasia or early malignant changes [33]. Leukoplakia is a clinical term used to describe a range of white oral lesions; it implies a diagnosis of exclusion of common conditions with similar appearance and harbors intrinsic potential malignancy [34–37]. Microscopically, these lesions are characterized by simple orthokeratosis, parakeratosis with epithelial hyperplasia and minimal inflammation, hyperkeratosis, or varying degrees of dysplasia. The latter occurs in up to 16% of leukoplakias [34]. Leukoplakias and erythroplakias (less frequent than leukoplakias in the general population) may undergo malignant transformations with or without clinical evidence of such change. Only 5–36% of white lesions can transform into malignancy within 20 years, the annual transformation rate of oral leukoplakia is unlikely to exceed 1%, and there is no proven correlation between transformation and the degree of dysplasia [38–41]. In spite of the progresses in molecular biology, there is not yet a single reliable marker predictive of malignant transformation [36, 37]. Clinically, early stages may be mistaken for reactive lesions that appear either as painless, nonhealing, indurated ulcerations, or hypertrophic lesions. Differential diagnosis is based on the analysis of the risk factors, the natural history, the progression and, most importantly, the clinical features of the lesion. A definitive diagnosis, however, can only be obtained after histological confirmation. Only then can the appropriate therapy be selected. The clinical conundrum for lesions without features of malignancy remains whether the initial biopsy is representative of the entire lesion, especially when they present with nonhomogeneous features [38, 42]. Microscopic foci of malignant tissue may be present and can only be detected histologically. Unexpected carcinomas in resection specimen have been reported for oral lesions removed after the initial incisional biopsy had not shown the presence of malignant tissue [38–43]. This lack of correlation between the histopathologic examination of

Conventional Treatment of Leukoplakias and Related Lesions In consideration of the reported malignant transformation rate of 5–36% [38–41], the therapeutic goal for oral leukoplakias is secondary prevention. Treatment modalities include lifestyle modification and elimination of risk factors, such as tobacco and alcohol intake, medical therapy with retinoids or antimycotics, surgical excision, cryosurgery, laser evaporation, or laser excision. Surgical excision is widely accepted to be the most effective form of treatment [36–44]. A useful initial approach in the management of oral leukoplakias should be the removal of etiologic factors in conjunction with simultaneous anti-inflammatory and antimycotic therapy. If clinical improvement or resolution is not obtained within a few weeks, surgical excision of persistent oral leukoplakias, preferably laser resection, seems to be the most rational next step [45]. However, results of prospective [46] and retrospective studies [36–45] describing rates of malignant transformation in patients treated with surgical or laser excision of oral leukoplakias are hardly comparable because of differences in diagnostic and inclusion criteria, followup time intervals, patient characteristics, and surgical techniques employed. The inconclusive data leaves unproven the hypothesis that surgical removal of potentially malignant oral lesions can prevent the onset of oral cancer [37, 38, 41, 47–49] and formed the basis for pilot chemoprevention studies.

Larynx Leukoplakias and Related Lesions Analogies exist between laryngeal and oral precancerous lesions: the presence of dysplasia has clinical relevance for both, but in laryngeal lesions a better correlation seems to exist between the grade of dysplasia and the clinical evolution of the lesion [50–54]. The natural history of untreated laryngeal dysplasia is well described for mild and moderate dysplasia. Invasive cancer can develop in as many as 45% of patients with moderate dysplasia and some authors have recommended intervention. For lesions with mild dysplasia, the rate of progression is reported to be up to 11.5% [49, 53].

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Conventional Treatment of Leukoplakias and Related Lesions As for the oral cavity, the management of premalignant lesions of the larynx is controversial. The best opportunity for cure must not be missed because of inadequate treatment and therapy must be oncologically radical with maximal functional preservation. The available data on the treatment of laryngeal premalignancy mostly addresses severe dysplasia/ carcinoma in situ [51–56]. A “wait-and-see” approach cannot be employed in these patients as some studies have indicated an unacceptably high rate of progression to invasive carcinoma. Intervention is recommended for all cases of severe dysplasia and/or carcinoma in situ [54]. Despite substantial recent advances there is significant morbidity associated with nonsurgical therapy sometimes used to treat these conditions [56] while laser surgery seems to be the best treatment modality to fulfill the requirements of oncologic radicality and organ as well as functional preservation [51, 52, 55].

Precancer and Risk Markers for Cancer A biological marker (biomarker) is a parameter that can be objectively measured and evaluated as an indicator of normal biological and pathogenic processes, gauging the response to therapeutic (most often pharmacological) interventions [57]. A small subset of biomarkers that demonstrate a strong correlation with the desired clinical endpoint can serve as its substitute. These surrogate endpoints are expected to be reasonably likely to predict clinical benefit or harm (or lack thereof) based on epidemiologic, therapeutic, physiopathologic, or other scientific evidence. The search for reliable biomarkers has an important impact on the evaluation of chemoprevention studies that goes beyond the potential changes to clinical practice. The evaluation of a marker linked to carcinogenesis requires the study of its expression in tumors; the presence of this marker (overexpressed, mutated, or masked) is analyzed in precancerous lesions or in normal tissue to assess if it is present as an indicator of a biologic process associated with the progression of a neoplasia [58]. In HNC chemoprevention trials, the search for reliable biomarkers focuses on identification of indicators of malignant transformation in clinically suspect lesions, those linked to second primary tumors and/or identification of individuals at greatest risk for the development of neoplasias [58]. SIN is defined as a noninvasive lesion with genetic abnormalities resulting in loss of cellular control functions with some phenotypic characteristics of invasive cancer [35, 59]. Preventive measures focus on evaluation and removal of its risk factors and surgical resection [45, 49, 51]. Epithelial tissues display SIN as moderate to severe dysplasia whose grade is determined by the degree of cellular abnormality above

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the epithelial basement membrane [34–38, 59]. Accuracy in grading is dependent on the quality of the tissue sample, the biopsy site, and the experience of the pathologist. Several studies have shown great inter- and intra-examiner variability in the assessment of presence, absence, and grade of oral epithelial dysplasia [35, 37, 59]. SIN is believed to represent (with appropriate sampling) the total field of abnormal epithelium and to provide identifiable lesions that can be targeted to evaluate the efficacy of new therapeutic interventions [28]. However, only a small portion of these lesions progress to cancer and they are not always indicative of malignant transformations [38, 42]. A striking discordance between the genetic status and the clinical and histologic features has been reported, particularly as it relates to treatment response [60]. Molecular studies also suggest that dysplasia may not be considered a reliable biomarker for cancer because high risk modifications can be found in nondysplastic lesions [49, 58]. There currently is not a body of evidence substantially strong enough to advocate in clinical practice the use of biomarkers as prognostic indicators for HNC [58]. Research in the field continues particularly with gene expression and salivary proteomics studies [61, 62] and recently published reports identify Podoplanin [63, 64] and the genotype CD1 AA and AG [54] as promising new markers.

Chemoprevention Chemoprevention is the use of natural or synthetic chemicals for the reversal, suppression, or prevention of conversion of a premalignant lesion to an invasive form [57]. In other words, chemoprevention includes all the interventions that employ agents aimed at preventing the development of cancer. Two basic concepts guide chemoprevention studies; the levels of acceptable toxicity must be much lower than in patients with cancers and the drug may only be administered orally [57, 65]. Premalignant lesions of the oral cavity represent an ideal model to study chemoprevention. Ready access allows easy monitoring and serial biopsies resulting in greater possibility of early intervention and faster data analysis [66, 67]. Only few studies have been conducted on laryngeal precancer because of limitations related to difficulty in access and monitoring [54, 68–70]. We can distinguish different forms of chemopreventive interventions: primary, adjuvant, and chemoprevention in high-risk population.

Primary Chemoprevention This form of chemoprevention includes treatment of precancerous lesions (leukoplakias) with agents acting to reverse morphological precursors of malignancy and to assess their efficacy.

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Table 2.1  Primary chemoprevention randomized trials Stich HF, 1988 [75] Design ß-Carotene 180 mg/week (Group I), ß-carotene + vitamin A 100,000 IU/week (Group II), placebo (Group III) Length of the study 6 months Patients included in the study 130 tobacco/betel chewers End points Complete remission (RC) of lkp and reduction of micronucleated cells Results Group I = 15%, Group II = 27% a, Group III = 3% Remarks Nobody changed the risk habits Stich HF, 1988 [76] Design Length of the study Patients included in the study End points Results Remarks Hong WK, 1986 [66] Design Length of the study Patients included in the study End points Results Remarks

Vitamin A 200,000 IU/week vs. placebo 6 months 54 tobacco/betel chewers Complete remission (RC) of lkp and prevention of new lkp Intervention group = 57% RC, 0% New lkp; Placebo = 3% RC, 21% New lkp Nobody changed the risk habits 13-cis-RA (1–2 mg/kg/day) vs. placebo 3-month intervention; follow-up = 6 months Intervention = 24; placebo = 20 Clinical = remission of leukoplakia; pathological = reversion of dysplasia Clinical-intervention group = 67%, placebo = 10%, p = 0.0002; pathological-intervention group = 54%, placebo = 10%; p = 0.01 Two severe toxicity; relapse of lkps in 56% of responding patients 2–3 months after intervention ended

Lippman SM, 1993 [77] Design

Phase I = 3-month high-dose 13-cis-RA (1.5 mg/kg/day) Phase II = 9-month low dose 13-cis-RA (0.5 mg/kg/day) (Group I); ß-carotene (30 mg/day) (Group II) Length of the study 12 months Patients included in the study Phase I (3 months) = 70; phase II (9 months) = 33 Group I, 26 Group II End points Remission of leukoplakia Results Phase I – remission of lkp = 36/66 (55%); progression of lkp = 7/66 Phase II – remission or stable disease 92% Group I vs. 45% Group II; p = 0.001 Group I = 8% progression, 1 Tis; Group II = 55% progression, 1 Tis, 5 SCC Remarks Mild, though greater toxicity in the high-dose 13-cis-RA evaluable patients after phase I (3 months) = 66; after phase II = 22 (Group I); 13 (Group II) lkp leukoplakia, is in situ carcinoma, SCC squamous cell carcinoma a  Statistically significant

Retinoids, ß-carotene, and a-tocoferol are the main agents employed in chemoprevention studies of oral leukoplakias. More than 30  years have elapsed since the initial clinical studies of natural vitamin A in the management of oral leukoplakia, and several single-arm studies have been reported [71–74]. Table  2.1 shows the design and the results of the published randomized trials [66, 75–77]. These studies demonstrate response rates that vary from 44 to 83% but revealed the dermatologic and liver toxicity of natural vitamin A. The effectiveness of these interventions is limited to the duration of the drug intake: a few weeks or months after stopping the drug intake the leukoplakias recur. Topical application of a natural or synthetic retinoid also achieved a temporary complete remission in more than 50% of patients, but the severe local side effects and the necessity to apply the drug locally limited this form of treatment and it is no longer used [45, 74]. Several authors conducted chemoprevention trials for laryngeal precancerous lesions [68, 69, 78]. The efficacy of the chemopreventive agents was less (clinical and histologic) than in oral lesions while similarities were noted in the overall

response profile (variability of response rate, side effects). Among these studies particular attention should be given to the Almadori trial [78]: a chemoprevention study with folates in patients with oral and laryngeal leukoplakias based on the observation that serum folate levels are significantly lower in patients with cancerous and precancerous lesions than in at risk and control patients. While there currently is no effective form of primary chemoprevention, its main role and goal remains to evaluate and test new agents that are effective and have a low sideeffect profile.

Adjuvant Chemoprevention: Prevention of Second Primary Tumors This form of chemoprevention consists of interventions on patients cured for HNC that employ a chemopreventive agent or a combination of agents in order to reduce the risk of second

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primaries. Patients treated for HNC have a constant and continuing risk of developing a second primary that varies from 2.7 to 4% yearly in the aerodigestive tract as well as in other sites [15, 16, 20, 79, 80]. Adjuvant chemoprevention might modulate epithelial cell biology and this way halt the progression of carcinogenesis [17, 81]. The development of synthetic vitamin A analogs (all-transretinoic acid, 13-cis-retinoic acid, etretinate, and phenretinide)

with potentially greater therapeutic indexes allowed the rapid expansion of chemoprevention trials [66, 77, 82]. Design and results of the published randomized trials are reported in Table 2.2 [67, 82–89]: in most of these the treatment regimens synthetic retinoids are taken alone or in association with ß-carotene. The reported protective effects are conflicting: in some studies retinoids seem to significantly reduce occurrence of second primaries [67, 82, 83], in others

Table 2.2  Adjuvant chemoprevention: results of the most significant randomized trials Hong WK, 1990 [82], Benner SE, 1994 [83] Design 13-cis-RA (50–100 mg/m2/day) for 12 months vs. placebo Length of the study Intervention = 12 months; follow-up = 54.5 months (median) Patients included in the study 103 disease-free patients after primary treatment for a HNSCC End point Occurrence of second primaries Results Intervention = 4%; placebo = 24%; p = 0.005 Remarks 13-cis-RA does not prevent recurrences and progression of the original tumor Difference in developing a second primary between treatment group diminishes in time, however, persists reduction of occurrence of second primary within head and neck area and lung Bolla M, 1994 [84] Design Length of the study Patients included in the study End point Results Remarks

van Zandwijk N, 2000 [85] Design Length of the study Patients included in the study End points Results Remarks

Etretinate (50 mg/day first month, and 25 mg/day 23 months) vs. placebo Intervention = 12 months; follow-up = 41 months (Median, range 0–81) 316 patients treated for T1/T2 N0/N1 £3 cm M0 HNSCC Occurrence of second primaries No differences between intervention group (28 second primaries) and placebo (29 second primaries) Multicentric study Treatment discontinued in 33% of patients due to toxicity vs. 23% in placebo, p = 0.05 Etretinate does not prevent recurrences and progression of the original tumor Group I – N-acetyl cysteine (600 mg/day/2 years); Group II – retinol palmitate (300,000 IU/day/1 year and 150,000 IU/day/1 year); Group III – both; Group IV – placebo Intervention = 24 months; follow-up = 49 months (Median) 2,595 patients treated for curable HNSCC (60%) and lung cancer (40%) Occurrence of second primaries and recurrences of the treated tumor No differences between the four groups Multicentric study 93.5% of patients have smoked; 25% continued to smoke after cancer diagnosis

Bairati I, 2005 [86], Meyer F, 2008 [87] Design a-Tocopherol (400 IU/day) and ß-carotene (30 mg/day) + RT vs. Placebo + RT Length of the study Intervention = 36 months; follow-up = 52 months (Median) Patients included in the study 400 with stage I–II HNSCC treated by radiation therapy End points Occurrence of second primaries and recurrences of the treated tumor Results a-Tocopherol higher risk than placebo of second primary (HR = 2.88) and recurrences (HR = 1.86) during the supplementation period, but lower rate when supplementation was discontinued (second primary HR = 0.41; recurrences HR = 0.33). Among smokers during RT highest risk of death for HNSCC (HR = 3.38) Remarks Multicentric study In the course of the trial ß-carotene was discontinued after 156 patients had enrolled because of ethical concerns Khuri FR, 2006 [88] Design Length of the study Patients included in the study End point Results Remarks

13-cis-RA (30 mg/m 2/day) vs. Placebo Intervention = 36 months, monitored for up to 4 years 1,190 early stage (I–II) HNSCC Occurrence of second primaries and overall survival No statistical difference Smoking statistically significantly increased the rate of second primary (HR = 1.64) and death (HR = 2.51) than nonsmoking (HR = 2.52) (continued)

2  Head and Neck Cancer Prevention Table 2.2  (continued) Perry CF, 2005 [89] Design Length of the study Patients included in the study End points Results Remarks

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Group I = high-dose isotretinoin (1 mg/kg/day for 1 year and 0.5 mg/kg/day for 2 years); Group II = moderate dose isotretinoin (0.5 mg/kg/day); placebo 3 years intervention 151 patients cured for a HNSCC Occurrence of second primary in head and neck are lung or bladder No significant difference in the occurrence of second primary, recurrence of primary disease or DFS Multicentric trial

Chiesa F, 2005 [67] Design Fenretinide 200 mg (1 year) vs. Placebo Length of the study Intervention = 12 months; follow-up = 60 months Patients included in the study 170 after resection of oral leukoplakia End points Recurrences of leukoplakias and occurrence of new leukoplakias and cancer Results Protective effect of fenretinide Remarks The protective effect lasted significantly for 7 months after drug interruption HNSCC head and neck squamous cell carcinoma, DFS disease-free survival

no protective effect was shown [84, 85, 88, 89]. The toxicity of etretinate is very high and many patients enrolled in the French study [84] discontinued treatment because of the side effects. The toxicity of high-dose isotretinoin was observed in all of the studies and its severity required many patients to discontinue therapy [83, 90–92]. On the contrary low-dose isotretinoin was well tolerated and was more effective than b-carotene. Several studies tested the effectiveness of another synthetic retinoid, N-(4-hydroxyphenyl) retinamide (fenretinide or 4-HPR) in preventing the clinical progression of oral leukoplakia via receptor-independent apoptosis and receptor-dependent effects [67, 93, 94]. These studies showed that fenretinide is a well-tolerated drug, able to prevent new occurrences of oral leukoplakias without improved efficacy at higher doses [93, 94]. After interruption of the pharmacotherapy, however, the protective effect of retinoids decreases over time and some patients can develop new leukoplakias and squamous cell carcinomas [66, 94]. In the Hong study [66, 83], the difference between the odds ratio of developing a second primary tumor at any site for isotretinoin-treated group diminishes over time and no statistically significant difference in survival has been observed. In the Chiesa study [67], the protective effect of fenretinide was shown to last significantly for 7  months after the completion of a 1-year intervention.

cancer of the upper aerodigestive tract because of lack of micronutrients and vitamin A in their diet, or of heavy alcohol and tobacco use. Intervention generally lasted several years and the results in term of reduced mortality from or reduced incidence of cancer were evaluated for at least 5 years after the end of the interventions. Table 2.3 shows the results of these trials [96–104]. Retinoid and micronutrient supplementation showed a protective effect in populations with low tissue ­levels of retinoids, but it was dangerous in individuals with normal retinoid levels, inducing a higher incidence of cardiovascular diseases and lung cancer. Two studies were stopped because of these results [98–103]. A relationship between lung cancer and serum ­levels of some carotenoids seem to show some gender predilection favoring males, with no apparent association observed among women [105]. These results and a critical review of the literature allow us to conclude that there is no evidence to support antioxidant supplementation for primary or secondary prevention, while Vitamin A, ß-carotene, and Vitamin E may increase mortality [106–108]. Future randomized trials could evaluate the potential effects of Vitamin C and selenium for primary and secondary prevention with close monitoring for potential harmful effects. Antioxidant supplements need to be considered medicinal products and should undergo sufficient evaluation before ­marketing [109].

Chemoprevention in High-Risk Populations

New Directions in Chemoprevention

This form of chemoprevention consists of dietary supplementation with vitamins, retinoids, and micronutrients in high-risk populations. During the final two decades of the last century several preventive studies have been conducted all over the world (China, Scandinavian countries, USA) [96, 97]. These trials included thousands of patients at risk for developing a

Following the conflicting and intriguing results of the early chemoprevention trials, other therapeutic regimens (single drug or combination) have recently been evaluated [95, 110–115]. Most studies tested anti-inflammatory drugs, including COXinhibitors and aspirin, because of the strong link between nonsteroidal anti-inflammatory drugs (NSAIDs) and the

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Table 2.3  Chemoprevention trials in high-risk populations Blot WJ, 1993 [96] Design Diet supplementation with Retinol + zinc (Group A), riboflavin + niacin (Group B), Vitamin C + molybdenum (Group C), ß-carotene + Vitamin E + selenium (Group D) Length of the study Diet supplementation = 5 years; follow-up = 2 more years Patients included in the study 29,584 at risk for esophageal and gastric cancer End point Decrease of mortality for esophageal, and stomach cancer Results Significantly lower mortality for cancer was found in group D, evident after 1–2 years Remarks No significant effect on mortality rates for all causes was found in the other arms Li JY, 1993 [97] Design Length of the study Patients included in the study End point Results Remarks

Diet supplementation with 12 minerals + 14 vitamins vs. placebo 6 years 3,318 subjects with esophageal dysplasia Decrease in cancer mortality and incidence No substantial short-term beneficial effect on incidence or mortality for esophageal cancer Cancer mortality 4% lower (RR = 0.96) and cerebrovascular disease 38% lower (RR = 0.62) in intervention group, not statistically significant

ABTC Study Group, 1994 [98], Albanes D, 1996 [99], Virtamo J, 2003 [100] Design a-Tocopherol (50 mg/day) (Group I), ß-carotene (20 mg/day) (Group II), both (Group III), placebo (Group IV) Length of the study 5–8 years (Median 6.1 years) Patients included in the study 29,153 Male Finnish, 50–69 years old, smokers ³5 cigarettes/day End point Reduction in lung cancer incidence Results Multicentric study Higher incidence of lung cancer and ischemic heart disease in those receiving ß-carotene No reduction in lung cancer in those receiving a-tocopherol Remarks Fewer prostate cancers, but more deaths from hemorrhagic stroke in the a-tocopherol group The beneficial and adverse effects of supplemental a-tocopherol and ß-carotene disappeared during postintervention follow-up Omenn GS, 1996 [101, 102], Goodman GE, 2004 [103] Design ß-Carotene 30 mg/day + Vitamin A 25,000 IU vs. Placebo Length of the study 4 years (stopped 21 months early than planned) Patients included in the study 18,314 smokers, former smokers and workers exposed to asbestos End point Decrease in lung cancer incidence Results Multicentric study Stopped due to higher incidence of lung cancers (RR = 1.28) and death for lung cancer (RR = 1.46) and for cardiovascular diseases (RR = 1.26) in the intervention group as compared with the placebo group The adverse effects persisted after supplementation was stopped (as of December 2004), although not statistically significant Lin J, 2009 [104] Design Length of the study Patients included in the study End point Results Remarks

Vitamin C Group (ascorbic acid 500 mg/day), vitamin E group (a-tocopherol 600 IU/every other day), ß-carotene group (50 mg/every other day), placebo 9.4 years (average) 7,627 women free of cancer before randomization Incidence and death from cancer No overall benefits in the primary prevention of total cancer incidence or cancer mortality Multicentric, double-blind, placebo-controlled 2 × 2 × 2 factorial trial

reduction of cancer incidence demonstrated in human ­epidemiological studies. The NSAIDs family inhibits the cyclooxygenase (COX) family of enzymes. COX-2 has been shown to be upregulated as much as 150-fold in HNSCC and 50-fold in the normal appearing tissue of patients with HNSCC compared with normal subjects [116]. However, problems and results of the first multicentric studies using these agents are similar to those obtained with the retinoids [117–120]. Heath et  al. [117] found that administration of 200 mg of celecoxib twice daily for 48 weeks of treatment

does not appear to prevent progression of Barrett’s dysplasia to cancer. In a hospital-based case-control study (529 patients with HNSCC vs. 529 controls), Jayaprakash et al. concluded that aspirin use reduces the risk of HNC (25%; OR 0.75) [119]. This effect is more pronounced in women and in individuals with low to moderate exposure to cigarette smoke or alcohol consumption. Heavy smokers and alcohol drinkers did not benefit from the protective effect of aspirin. Current basic science advances are swiftly followed by an inability to translate them into clinically relevant interventions,

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to verify end-points and to establish adequate follow-up. As of September 2009 the National Institute of Health [120] reports six recruiting chemoprevention clinical trials using molecular agents (kinase or serin protease inhibitors), and antiinflammatory drugs (COX-2 inhibitors, sulindac, or acetyl salicylic acid) as single agents or in combination. In addition to these, 11 other primary or adjuvant chemoprevention trials are currently active, but not yet in the recruiting phase: their purpose is to test the effectiveness of natural and synthetic retinoids (four trials), dietary supplementation (one study), anti-inflammatory (four studies), and antidiabetic drugs (two studies). Chemoprevention trials are expensive because of the large study population needed and the necessary length of the studies. Cost analysis of these trials includes the sample size, the total number of study subjects and the necessary lengthy follow-up, the number of trial outcomes evaluated, possible delays in the accrual process, and cost effectiveness of particular retention activities. Based on the negative experiences made with the CARET study, the psychological effects of information relating to possible negative outcomes of the study (involving healthy population) should also be considered [121, 122]. The original promise HNC chemoprevention will be fulfilled only if putative biomarkers are validated with well designed and adequately funded long-term studies, that allow the creation of accurate molecular risk stratification models and translate into significant changes to clinical practice [17, 81, 93, 123, 124].

Prevention of Neck Metastases One of the basic issues of secondary and tertiary prevention in HNC is linked to the possibility of prevention of neck metastases [125, 126]. The neck is the central point in the management of HNSCCs; once metastases become clinically apparent, extra-capsular spread (ECS), a known prognostic factor [125, 126, 132], is more likely than in occult metastatic disease where ECS is estimated to be more than 15–20% [126–128]. ECS does not depend on the quantity of tumor cells present in the metastatic nodes: up to 60% of micrometastatic nodes (cN0 pN1) show ECS [129–131]. A study by Woolgar evaluating the treatment results in a series of patients with head and neck cancer [128] showed that, regardless of the T stage, the overall survival (OS) depends upon the pathological lymph node status: OS in pN0 patients was 73%, in pN+ without ECS = 51%, and in pN+ with ECS was 29%. Distant metastases and local recurrences are also significantly related to the lymph node status [132]. Currently, the only specific predictive factors of lymph node metastases are the site, size, and thickness of the primary tumor [130, 133].

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Identification of factors affecting invasion and metastasis, as well as the establishment of biomarkers to predict malignant potential and to identify different risk groups are of paramount importance. Cancer cell invasion and metastasis are a complex, multistep process involving interactions between invading cells, the extracellular matrix, and other stromal elements. In the initial phases of tumor progression, tumor cells undergo genetic changes, providing proliferative advantages such as the ability to resist growth-inhibiting signals, avoidance of programed cell death (apoptosis), induction of blood vessel growth (angiogenesis), loss of cell adhesion and migration, lymphatic angiogenesis, and the ability to survive in the environment of the metastatic site [134–136]. When these metastatic capacities are acquired (early or late) in tumor progression remains unclear, however there is evidence suggesting, in contrast to common belief, early acquisition of this transformation [136]. Many of these competitive advantages may also vary in time. For example, cell adhesion should decrease to allow cells to migrate and metastasize, but cell adhesion is again needed to settle at the metastatic site [134].

Search for Additional More Sensitive Markers Many biomarkers have been studied to establish correlation with the presence of nodal metastases in HNSCC with widely varying results and include matrix-metallo proteinases (MMP) [134, 137–145], podoplanin [63, 64], p27, ki-67 [146–148], and vascular endothelial growth factor (VEGF) [149, 150]. Recently, new techniques centered upon gene-expression profiling and comparative genomic hybridization with microarray technology have been developed and have allowed reliable detection of predictors of behavior rather than single markers [134, 151–156]. The findings of these studies indicate that these markers identify a subset of patients with poor prognosis, requiring aggressive treatment modalities, including new molecular targeted therapies likely to act as anti-invasion and antimetastatic therapeutic agents [157].

HPV Infection The HPV is part of a very heterogeneous family of viruses. It represents an important human carcinogen, causing the vast majority of cervical and anogenital tumors, and a variable number of cancers in other districts of the human body including the head and neck [158, 159]. HPV-positive SCCHN have been reported to share some epidemiological and biological characteristics with anogenital carcinomas [160, 161].

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A  clear geographic variability in cancer risk and burden Risk Factors for HPV Infection, Oral, and Oropharyngeal Squamous Cell Carcinomas exists across countries and specific interventions are required HPV infection is thought to precede the development of an HPV-positive HNSCC. The presence of high-risk HPV infection in oral mucosa and seropositivity increases significantly the risk of development OSCC [162–166]. Therefore, risk factors for HPV oral infection are likely, by extension, to be risk factors for HPV-positive HNSCC. Patients with HPVpositive tumors appear to be distinct from HPV-negative patients. There is no gender predilection, patients are often nonsmokers and nondrinkers [167, 168] and younger than HPV-negative tumors [169]. The degree to which oral HPV infection may combine with tobacco and/or alcohol use to increase risk of cancer is unclear [160, 169]. In the majority of the studies OSCC related to HPV infection have a better outcome and a reduced risk of relapse and second tumors as compared with HPV-negative tumors [160, 170, 171].

Vaccination as a Form of Prevention Vaccines designed strictly for prevention of cervical cancer and vulvar genital warts have recently been introduced. The existing vaccines are able to create a robust humoral immune response [172, 173] that is much more effective than the levels of antibodies acquired after a natural infection, and persist at least for a 60-month period [172]. Five-year follow-up demonstrates 100% effectiveness in prevention of persisting infection as well as HPV-16 and HPV-18 CIN 2/3 lesions in young women [173]. HPV-16 is found in the majority of HPV-positive oral cancer [173]. All vaccine trials reported to date have been designed to investigate the ability to generate protection against anogenital HPV infection in women. There is reason to believe that the existing vaccines may be effective against oral HPV infection, and prevent vaccine-type HPV-related HNC in both men and women [172, 174]. Data also suggests that therapeutic vaccines are effective against low-volume disease and could be used as adjuvant therapy following surgery or radiotherapy to clear microscopic residual disease [4]. Clinical trials to evaluate the efficacy of the quadrivalent HPV vaccine (against HPV 6, 11, 16, 18) in protecting against oral infection are currently being developed.

in each region. Primary prevention is considered the best form of prevention. Implementation of a primary prevention program requires knowledge of the specific risk factors (tobacco, alcohol, HPV infection) and the ability to limit exposure and to remove them. Efforts to promote healthy lifestyle practices such as tobacco control and cessation programs, recommendation for dietary modification (including alcohol consumption reduction) and weight control have yielded mixed results without significant reduction in the incidence of new cases of HNSCC [41, 175]. This observation highlights the fact that achieving primary prevention is very difficult and has given greater relevance to secondary prevention. Early detection and diagnosis entails by definition the discovery of preneoplastic lesions and early carcinomas. Precancerous lesions and cancer are part of a clinical continuum making it difficult to define where one ends and the other begins. Consequently, it becomes difficult to definitively state what represents therapy for one end of the disease spectrum versus the other [157, 176]. Genetic aberrations do not always result in visible lesions and a large portion of all preneoplastic lesions remains clinically silent. Even recognizing preneoplastic alterations, currently there is no sufficient evidence suggesting that the surgical treatment of precancerous lesions reduces the incidence of cancer [41]. The rapid development of molecular biology, the identification of the fundamental cancer genes and signaling pathways, and the development of new functional diagnostic imaging techniques show renewed promise for early prevention. The stratification of patients in different subgroups based on etiology, genomic classification, and other parameters clearly has important implications. Other than showing promise, however, we have not been able to translate this new knowledge into clinically successful strategies for early detection or chemoprevention of cancer. We are again at the dawn of a new era with the conclusion of the Human Genome Sequencing Project and advances in molecular and cellular pathophysiology hold yet more promise that a deeper understanding of the fundamental disease mechanisms may result in improved prevention and cure. The challenges remain in the correct interpretation of these findings and in their wise and scientific application. Only then will we be able to impact the field of HNC, transforming prevention into the only form of cure.

References Conclusions Improvement in the field of prevention requires a multidisciplinary approach. The development of cancer is a complex process, and multiple factors may be crucial in prevention.

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F. Chiesa et al. 137. MacDougall JR, Matrisian LM. Contributions of tumor and stromal matrix metalloproteinases to tumor progression, invasion and metastasis. Cancer Metastasis Rev. 1995;14:351–62. 138. Rosenthal EL, Matrisian LM. Matrix metalloproteases in head and neck cancer. Head Neck. 2006;28:639–48. 139. Rosenthal EL, Johnson TM, Allen ED, Apel IJ, Punturieri A, Weiss SJ. Role of the plasminogen activator and matrix metalloproteinase systems in epidermal growth factor- and scatter factorstimulated invasion of carcinoma cells. Cancer Res. 1998;58: 5221–30. 140. O-charoenrat P, Sarkaria I, Talbot GS, Reddy P, Dao S, Ngai I, et al. SCCRO (DCUN1D1) induces extracellular matrix invasion by activating matrix metalloproteinase 2. Clin Cancer Res. 2008;14:6780–9. 141. O-charoenrat P, Rhys-Evans PH, Eccles S. Expression of matrix metalloproteinases and their inhibitors correlates with invasion and metastasis in squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg. 2001;127:813–20. 142. Peterson JT. Matrix metalloproteinase inhibitor development and the remodeling of drug discovery. Heart Fail Rev. 2004;9:63–79. 143. Mclntyre JO, Fingleton B, Wells KS, Piston DW, Lynch CC, Gautam S, et  al. Development of a novel fluorogenic proteolytic beacon for in  vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity. Biochem J. 2004;377:617–28. 144. Bertini I, Calderone V, Cosenza M, Fragai M, Lee YM, Luchinat C, et al. Conformational variability of matrix metalloproteinases: beyond a single 3D structure. Proc Natl Acad Sci USA. 2005;102:5334–9. 145. Okada A, Bellocq JP, Rouyer N, Chenard MP, Rio MC, Chambon P, et  al. Membrane-type matrix metalloproteinase (MT-MMP) gene is expressed in stromal cells of human colon, breast, and head and neck carcinomas. Proc Natl Acad Sci USA. 1995;92:2730–4. 146. Thomson PJ, Hamadah O, Goodson ML, Cragg N, Booth C. Predicitng recurrences after oral precancer treatment: use of cell cycle analysis. Br J Maxillofac Surg. 2008;46:370–5. Epub 2008 Feb 20. 147. Pignataro L, Sambataro G, Pagani D, Pruneri G. Clinico-prognostic value of D-type cyclins and p27 in laryngeal cancer patients: a review. Acta Otolaryngol Ital. 2005;25:75–85. 148. Lothaire P, de Azambuja E, Dequanter D, Lalami Y, Sotiriou C, Andry G, et al. Molecular markers of head and neck squamous cell carcinoma: promising signs in need of propsective evaluation. Head Neck. 2006;28:256–69. 149. Joo KH, Jung CK, Kim MS, Sun DI. Relationship between vascular endothelial growth factor and Notch1 expression and lymphatic metastasis in tongue cancer. Otolaryngol Head Neck Surg. 2009;140:512–8. 150. Boonkitticharoen V, Kulapaditharom B, Leopairut J, Kraiphibul P, Larbcharoensub N, Cheewaruangroj W, et al. Vascular endothelial growth factor a and proliferation marker in prediction of lymph node metastasis in oral and pharyngeal squamous cell carcinoma. Arch Otolaryngol Head Neck Surg. 2008;134:1305–11. 151. Agra IM, Carvalho AL, Pinto CA, Martins EP, Filho JG, Soares FA, et al. Biological markers and prognosis in recurrent oral cancer after salvage surgery. Arch Otolaryngol Head Neck Surg. 2008;134:743–9. 152. Hensen EF, De Herdt MJ, Goeman JJ, Oosting J, Smit VT, Cornelisse CJ, et al. Gene-expression of metastasized versus nonmetastasized primary head and neck squamous cell carcinoma: a pathway-based analysis. BMC Cancer. 2008;8:168–78. 153. Chen ZG. Exploration of metastasis-related proteins as biomarkers and therapeutic targets in the treatment of head and neck cancer. Curr Cancer Drug Targets. 2007;7:613–22. 154. Yang MH, Chang SY, Chiou SH, Liu CJ, Chi CW, Chen PM, et al. Oncogene. 2007;26:1459–67. Epub 2006 Aug 28.

2  Head and Neck Cancer Prevention 155. Cromer A, Charles A, Millon R, Ganguli G, Chalmel F, Lemaine F, et al. Identification of genes associated with tumorigenesis and metastasic potential of hypopharyngeal cancer by microarray analysis. Oncogene. 2004;23:2484–98. 156. Carinci F, Lo Muzio L, Piattelli A, Rubini C, Chiesa F, Ionna F, et al. Potential markers of tongue tumor progression selected by cDNA microarray. Int J Immunopathol Pharmacol. 2005; 18:513–24. 157. Lippman SM, Heymach JV. The convergent development of molecular-targeted drugs for cancer treatment and prevention. Clin Can Res. 2007;13:4035–41. 158. Walboomers JM, Jacobs MV, Manos MM. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999;189:12–9. 159. Finch JT, Klug A. Papillomaviruses in the causation of human cancers – a brief historical account. Virology. 2009;384(2):260–5. Epub 2009 Jan 8. 160. Herrero R, Castellsague X, Pawlita M. Human papillomavirus and oral cancer: the International Agency for Research on Cancer multicenter study. J Natl Cancer Inst. 2003;95:1772–83. 161. Gillison ML, Koch WM, Capone RB. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst. 2000;92:709–20. 162. Smith EM, Ritchie JM, Summersgill KF. Age, sexual behavior and human papillomavirus infection in oral cavity and oropharyngeal cancers. Int J Cancer. 2004;108:766–72. 163. Hansson BG, Rosenquist K, Antonsson A. Strong association between infection with human papillomavirus and oral and oropharyngeal squamous cell carcinoma: a population-based case-control study in southern Sweden. Acta Otolaryngol. 2005;125:1337–44. 164. Mork J, Lie AK, Glattre E. Human papillomavirus infection as a risk factor for squamous-cell carcinoma of the head and neck. N Engl J Med. 2001;344:1125–31. 165. D’Souza G. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med. 2007;356:1944–56.

55 166. Smith EM, Ritchie JM, Pawlita M, Rubenstein LM, Haugen TH, et al. Human papillomavirus seropositivity and risks of head and neck cancer. Int J Cancer. 2007;120:825–32. 167. Weinberger PM. Molecular classification identifies a subset of human papillomavirus-associated oropharyngeal cancers with favourable prognosis. J Clin Oncol. 2006;24:736–47. 168. Gillison ML. Human papillomavirus and prognosis of oropharyngeal squamous cell carcinoma: implications for clinical research in head and neck cancers. J Clin Oncol. 2006;24:5623–5. 169. Lindel K, Beer KT, Laissue J. Human papillomavirus positive squamous cell carcinoma of the oropharynx: a radiosensitive subgroup of head and neck carcinoma. Cancer. 2001;92:805–13. 170. Fakhry C, Westra WH, Li S. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst. 2008;100:261–9. 171. Nichols AC, Faquin WC, Westra WH, Mroz EA, Begum S, et al. HPV16 infection predicts treatment outcome in oropharyngeal squamous cell carcinoma. Otolaryngol Head Neck Surg. 2009;140:228–34. 172. Harper DM, Franco EL, Wheeler CM. Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial. Lancet. 2006;367:1247–55. 173. Mao C, Koutsky LA, Ault KA. Efficacy of human papillomavirus-16 vaccine to prevent cervical intraepithelial neoplasia: a randomized controlled trial. Obstet Gynecol. 2006;107:18–27. 174. Reisinger K, Block SL, Lazcano-Ponce E. Safety and persistent immunogenicity of a quadrivalent vaccine in preadolescents and adolescents: a randomized controlled trial. Pediatr Infect Dis J. 2007;26:201–9. 175. Greenwald P. The future of cancer prevention. Semin Oncol Nurs. 2005;21:296–8. 176. Abbruzzese JL, Lippman SM. The convergence of cancer prevention and therapy in early-phase clinical drug development. Cancer Cell. 2004;6:321–6.

Chapter 3

Cellular and Molecular Pathology of Head and Neck Tumors Adel K. El-Naggar

Abstract  Head and neck pathology encompasses a multitude of organs of diverse histogenesis. Malignances arising from head and neck sites accordingly are diverse in origins, morphogenesis, and biological behavior. Excluding connective tissue and vascular entities, the main entities that are presented in this chapter include squamous mucosal sites, salivary, thyroid and sinonasal, and skull base tumors. The histopathological classification remains the main reference to the diagnosis and to a large extent, malignancy grading. Advances in immunohistochemical techniques and the development of reagents to cellular intermediate filaments and lineage markers have led to better diagnosis and categorization of undifferentiated entities with overlapping morphologic features. More recently, major strides have been achieved in the molecular genetic characterization and understanding of head and neck tumorigenesis. Although clinically applicable and validated molecular biomarkers have yet to be realized, it is important to address the recent discoveries and their potential integration with the phenotypic and pathologic features. This chapter concisely presents the relevant pathomorphologic and molecular features of the tumors of the major head and neck sites for clinical management. Keywords  Head and neck squamous carcinoma • Molecular genetics • Squamous tumorigenesis • Tumor heterogeneity

lining of the upper respiratory tract mainly in individuals with a history of abusing risk factors, including cigarette smoking, alcohol abuse, and human papillomavirus. Only 20% of individuals with these risk factors, however, develop squamous carcinoma [1, 2]. Head and neck mucosal sites are an ideal model of investigating the molecular genetic alterations leading to squamous carcinoma development because of their readily accessible location, association with known risk factors, and the presence of defined histopathologic progression stages. In contrast to other major cancer types, HNSC lacks familial inheritance, is difficult to cultivate and there are no faithful animal models to advance research and development in this field [1]. Squamous tumorigenesis is thought to result from successive accumulation of molecular genetic alterations in the squamous epithelium lining the upper aerodigestive tract [1, 2]. Although the temporal occurrence and the order of these events are largely unknown, some certainly precede the phenotypic changes associated with preinvasive dysplastic lesions. The progression of late stage dysplasia to invasive carcinoma is a complex one and comprised of both cellular and structural changes as a result of dysregulation of key pathways triggered by interaction of epithelium and the host stromal elements [3] (Fig. 3.1).

Histopathology Squamous Mucosal Carcinogenesis Head and neck squamous carcinoma (HNSC) is the fifth most common cancer worldwide with approximately 500,000 new cases per year. They develop from the squamous mucosal

The diagnosis and management of head and neck mucosal lesions are based on the histopathologic assessment of biopsied or excised specimens.

Oral Premalignant Lesions A.K. El-Naggar (*) Department of Pathology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Unit 085, Houston, TX 77030, USA e-mail: [email protected]

These lesions are recognized as grossly abnormal mucosa of no definitive etiology and can broadly be classified into leukoplakia (white) and erythroplakia (red). The risk of developing

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A.K. El-Naggar

Fig. 3.1  Phenotypic and molecular progression model of head and neck squamous tumorigenesis

invasive carcinoma from these lesions varies greatly and range from 3 to 16% for leukoplakia and from 30 to 50% for erythroplakia [2].

sive carcinoma (>3 mm) are generally treated with complete excision without neck dissection. Lesions with more than 5 mm invasion are eligible for neck dissection [2].

Leukoplakia

Squamous Carcinoma Variants

Leukoplakia is defined as a persistent white area of unknown etiology. These lesions may present as either discrete homogenous or delimited nonhomogenous forms. Generally, the nonhomogenous lesions are associated with higher risk than their homogenous counterparts. The majority of leukoplakias develop in tobacco consuming individuals and their location and appearance varies according to the geographic location and the manner and nature of the tobacco consumption. A definitive diagnosis is based on the histopathological evaluation of lesional biopsy and serves to rule out mimics, such as Lichen Planus and to assess the presence or absence of dysplasia [2]. Histologically, leukoplakia is characterized by epithelial hyperplasia with hyperkeratosis and/or parakeratosis. The development of dysplasia in these lesions is heralded by progressive alteration of the squamous epithelium manifested by changes in basal cell polarity and cellular and nuclear features and is graded as mild, moderate, or severe based on the extent of the dysplastic cellular features.

Squamous carcinoma manifests multiple, distinct phenotypes with variable site predilections and biological behaviors and include verrucous, papillary, basaloid, and sarcomatoid phenotypes [5].

Verrucous Hyperplasia Verrucous hyperplasia grossly appears as a white, warty raised growth mainly in the oral cavity. Both verrucous hyperplasia and carcinoma share clinically and pathologically similar and overlapping features. Verrucous hyperplasia shows exophytic growth with minimal inward stromal involvement. A diagnosis can only be achieved by an excisional biopsy where the edges and the full depth of the lesion are represented. The histologic diagnosis, therefore, is generally arbitrary and the difference is essentially academic since both lesions should be completely excised [4].

Erythroplakia Erythroplakia is defined as a grossly red squamous mucosa. They present as either homogenous or nonhomogenous red mucosa with and without leukoplakia association. Erythroplakia represents the end stage of dysplasia histologically and carries the highest risk of progression to invasive squamous carcinoma. Both severe dysplasia and microinva-

Verrucous Carcinoma This is a locally invasive squamous carcinoma with warty gross features and minimal cellular abnormalities. These lesions may frequently present in oral and laryngeal sites and in its pure form, has minimal metastatic potential.

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Verrucous carcinoma typically affects the oral and laryngeal sites, is locally invasive and in pure form, rarely metastasizes. Histologically, these tumors are well differentiated and invade with broad pushing borders [5].

Viral Associated Squamous Carcinoma Subtypes

Conventional Squamous Carcinoma

Increasing evidence links HPV as an etiologic agent in the development of a subset of HNSC. Current data indicate that the majority of these cases are oropharyngeal, including the tonsils. This is further supported by the high risk of oropharyngeal carcinoma in seropositive HPV-16 and high risk of anogenital cancer patients. The exact prevalence of HPV in HNSC is not accurately known with figures ranging from 5 to >70%. These variations are related to several factors, including differences in population, tumor sites, method of HPV detection, and histological subtypes. It is clear, however, that HPV-16 is dominantly present in more than 50% of patients with oropharyngeal SCC. Integration of viral DNA into the nuclear genome is a critical step in the malignant transformation. Subsequent to viral integration, detection of early genes (E2) occurs and upregulation of E6 and E7 genes is noted. The E6 of the HPV-16 bind to the p53 suppressor genes, consequently, and lead to uncontrolled proliferation of the oropharyngeal squamous mucosa. It has also been shown that elevated expression of p16 is a surrogate marker in HPV infection. Approximately 10–60%, dependent on the population and the site of infection of HNSCs, are reported to harbor HPV. Patients with this type of tumor respond better, do not have traditional risk factors, and have better survival. E7 leads to inactivation of Rb protein and the release of the transcription factor E2F and the upregulation of both p14 and p16 proteins. Evidence for viral integration, especially in tonsillar carcinoma, in tumor cells is critical to the diagnosis. Also, the detection of p16 overexpression as an alternative/complimentary to the detection of HPV infection may be helpful. The contribution of viral load to variations in reporting these markers remains to be addressed. In one study, high viral load of <60  copies/cells was found to correlate positively with survival; however, a later subsequent larger study failed to confirm this finding [6, 8–13]. The traditional risk factors associated with conventional squamous carcinoma may play a secondary, but deleterious role in this demographic population. Only certain oncogenic subtypes of the papilloma virus, especially HPV-16 and l8, have been identified as etiologic factors in tumorigenesis of HNSC. The E6 and E7 genes of the HPV-16 genes bind to the p53 and Rb suppressor genes and upregulation of the p16-IK4 inhibitor leading to dysregulation of the cell cycle and tumor development. Interestingly, these tumors are less aggressive and more sensitive to conventional therapy than conventional squamous carcinoma.

This is the most common form of presentation and typically graded based on the degree of squamous epithelial alterations and state of keratinization into well, moderately and poorly differentiated carcinoma. The pattern of invasion of these lesions may also impact on the extent of invasion, metastasis, vascular, and perineural permeation. Generally, broad, invasive fronts are less ominous than finger-like invasive fronts [1]. Papillary Squamous Carcinoma Papillary squamous carcinoma is typically laryngeal or nasal in origin and is exophytic in presentation with minimal tissue invasion. An association with HPV infection has been suggested, but remains uncertain. Papillary squamous carcinoma typically pursues less aggressive behavior than the other forms of squamous carcinoma, except the verrucous variant [5]. Basaloid Squamous Carcinoma This is a unique high-grade variant of squamous carcinoma with a predilection for hypopharyngeal, tonsillar, and base of tongue sites. They are characterized by uniform, highly malignant basaloid cells with focal squamous differentiation and collagen-like deposition. Recently, an association with high-risk HPV infection has been reported. Morphologically, tumors are characterized by a proliferation of homogenous basaloid cells with necrosis and focal abrupt areas of luteinization. These tumors may be confused with solid adenoid cystic and neuroendocrine carcinomas [5, 6]. Sarcomatoid Squamous Carcinoma Two forms of sarcomatoid squamous carcinoma are recognized: the exophytic form and the ulcerative invasive forms. The exophytic are usually found in laryngeal and hypopharyngeal sites and may or may not manifest areas of conventional squamous carcinoma. The distinction between this entity and pure sarcoma is based on combined morphologic and immunohistochemical staining for keratin intermediate filaments. Patients with the exophytic form may pursue a relatively better clinical course than the endophytic counterpart [7].

Oropharyngeal Carcinoma

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Nasopharyngeal Carcinoma This is a unique form of HNSC that develops in the nasopharyngeal region. They are classified based on their histological appearance into differentiated squamous carcinoma (WHO I) and undifferentiated carcinoma with lymphoid stroma (WHO II or III). The histologic features of type I are similar to well-differentiated squamous carcinoma, while the types II and III are highly undifferen­ tiated carcinoma with integral lymphoid components. Nasopharyngeal carcinoma (NPC) is associated with Epstein–Barr virus infection especially in patients from the Orient and Middle East but less likely in patients from the Western hemisphere. These tumors are highly sensitive to radiation therapy [14].

Adverse Pathologic Features of Clinical Relevance The following histopathologic factors are considered features associated with high risk of recurrence and failure to therapy response: 1 . Poor histologic differentiation 2. Finger-like and single cell invasive pattern 3. Perineural invasion 4. Close surgical margins (<5 mm) 5. Presence of high-grade dysplasia 6. Extra nodal extension of lymph node metastasis [15]

Molecular Pathology Cellular Concept The molecular and biological analysis and understanding of squamous tumorigenesis of the head and neck is largely based on the concept of field characterization conceived by Slaughter et al. in 1953 [16]. This concept assumes that risk factors render the entire aerodigestive mucosal surface susceptible to the squamous carcinoma development. In the small subset of patients with no history of risk factors, and/ or short temporal exposure to these factors, an inherent genetic susceptibility may play a role [1, 17, 18]. The cellular concept’s premise for squamous carcinoma development and progress is that HNSC carcinoma results from molecular and/or biological alterations in the squamous epithelial cells.

A.K. El-Naggar

DNA-Based Studies LOH Findings Microsatellites are short tandem repeat DNA sequences scattered throughout the genome. The vast majority of these repeats are polymorphic, inherited differently from each parent among different populations. Using constitutional DNA extracted from fresh or archived specimens as a standard, loss or shift in mobility in tumor microsatellite bands on gel electrophoresis, determines the presence or lack of microsatellites of alterations. In general, frequent loss of loci on chromosomes 3p, 9p, and 17p has been detected in premalignant squamous lesions and many constitute any early alterations that may be used in screening of high risk individuals for early detection of cancer. Other chromosomal alterations, including 4q, 6p, 8p, 11q, 13q, and 18q are typically more frequent in invasive and advanced squamous carcinomas. Chromosomal gains, in contrast, are infrequent in squamous tumorigenesis and limited to chromosomes 3q26 and 11q13 amplicons and generally are late events [18–22]. Specific Gene Findings p53 gene: p53 is a tumor suppressor gene on chromosome 17p. It is the most frequently mutated gene in HNSC in approximately 50% of the cases. Tumors from patients with long histories of risk factor exposure are more frequently mutated. Most of the p53 mutations are transversion in type (G:T), but missense mutations can also be found and clustered between exons 5 and 9. p16 gene: p16 is another tumor suppressor gene on chromosome 9p21. Loss of p16, a potent inhibitor of cell cycle, leads to uncontrolled proliferation. In contrast to p53, mutations of p16 are infrequent events in HNSC. Instead, hypermethylation of the p16 promoter and the first exon is the major mechanism for loss of function [23–25]. FHIT gene (Fragile histidine triad): FHIT, on the short area of chromosome 3p14.2, has also been implicated in HNSC. However, the frequency and the temporal involvement of this gene in squamous tumorigenesis remain undefined [2]. Cyclin-D1 gene: Cyclin-D1, a critical cell cycle gene within chromosome 11p amplicon, has also been found to be highly amplified in advanced premalignant and invasive lesions. Polymorphism at this gene has been associated with high risk of developing squamous carcinoma [26]. p63 gene: P63 is a member of the p53 gene family and located on chromosome 3q29-29 region. P63 is a vital gene in normal epithelial development and has been implicated in

3  Cellular and Molecular Pathology of Head and Neck Tumors

several epithelial tumor developments. P63 has two different promoters resulting in two different protein products, on retaining the transactivation domain (TA p63) and another lacking it (D(delta)Np63). Both isotypes undergo alternative splicing at the carboxy terminal leading to six isoforms (three each) (a(alpha), b(beta), and n(upsilon)). Studies of this gene and its main isotypes in HNSC indicate an important role in tumorigenesis, especially the DN isotypes. Overexpression of this isotype blocks differentiation and metastasis, promotes proliferation in HNS tumorigenesis, and may be an attractive target for therapeutic intervention in a subset of patients with these tumors [27, 28].

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on the extent of base pairing with target RNA, miRNA may lead to translational regression of degradation. Because of the partial complementarity between miRNAs and their targets, each miRNA may regulate several genes. A few recent studies of these molecules have recently been published. Several miRNAs, including miR-375 and miR-221, have been found to be significantly altered in HNSC [35, 36]. Another study of squamous carcinoma of the tongue identified 24 upregulated and 13 downregulated miRNAs. Of the most significantly upregulated, miR-184 was identified. Inhibition of the miR-184 cell lines led to decreased proliferation, downregulation of C-Myc, and induction of ­apoptosis. Further analysis of these molecules is warranted for their potential therapeutic use [37].

Epigenetic Alterations

Growth Factors and Signal Transduction Epigenetic alteration is the process of gene silencing by non- Pathways DNA alterations and includes cytosine methylation of the CpG islands at the promoter and/or chromatin modulation and histone acetylation. These epigenetic modifications are reversible and may be of future therapeutic value. Cytosine methylation of several tumor suppressor genes in HNSC has been the target of numerous studies. Genes that have been found to be highly methylated in HNSC include p16, MGMT, RARB, E-Cadherin, and DAPk [29, 30]. The diagnostic and therapeutic potential of these alterations remain to be achieved.

Genomic Studies In genomic studies of HNSC using varied platforms, patient populations have recently been conducted. The inherent heterogeneity of these tumors complicates the interpretation and renders a clear conclusion difficult. Although results have shown evidence for segregating different responsive and aggressive behaviors, lymph node metastasis and tumor sites, the complexity of the analysis and the heterogeneity of tumors and biological behaviors limit the clinical utilization of these platforms [31–34].

MicroRNAs MicroRNAs, highly conserved and ubiquitous short (18–22 nt) noncoding RNA sequences, were found to regulate gene expression posttranscriptionally by base pairing with 3¢-UTR (untranslated region) of cognate RNA transcript. Dependent

Understanding the signaling pathways, trafficking and regulation of fundamental, inter- and intracellular tumor/host interactions, will lead to understanding the biology of individual tumors and the development of effective targeted therapy in HNSC. Alterations in several growth factor receptor pathways play a critical role in the development and progression of HNSC. Several growth factors affecting signaling pathways in HNSC have been identified. These include the EGFR, Ras, NFkB, TGFb, and PI3k/AkT/mammalian target of rapamycin (mTOR) pathways.

Epidermal Growth Factor The epidermal growth factor (EGFR) gene is located on the short arm of chromosome 17 and encodes for a transmembrane tyrosine-kinase receptor expressed on several epithelial cells. EGFR activation is a critical early event in the development of squamous carcinoma. EGFR is a glycoprotein receptor with a cystin-rich ligand-binding domain with short sequence and intracellular tyrosine kinase and carboxy-terminal scaffolding domains. The activation of EGFR family members is either through ligand dependence or independence. The independent activation is the result of mutation or overexpression-induced homodimerization or heterodimerization with other Grb family members. Ligand-independent activation of EGFR in HNSC has been linked to a transi­ tion mutation, EGFR variant III (EGFRvIII). Ligand binding to the EGFR initiates phosphorylation and triggers a signal transduction cascade that result in the activation of

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A.K. El-Naggar

Fig. 3.2  Proposed model of epithelial to mesenchymal transition in squamous tumors

downstream molecules and increase in cell proliferation. Overexpression of EGFR has been amply reported in HNSC to be associated with aggressive behavior, poor progression and response to targeted anti-EGFR therapy [38–40]. Studies of mutations in the hot spot exons of this gene have yielded negative results. However, increased gene copy numbers have been reported in a subset of these tumors. Currently, immunohistochemical staining with anti-EGFR is the most commonly used method of assessment of this gene. It is unknown, however, whether the activated form (phosphorylated) or the total EGFR level correlates better with the activity and response to therapy in HNSC [41]. The interest and available data on EGFR have led to interest in the development of molecularly targeted small-molecule inhibitors in the treatment of HNSC. New anti-EGFR tyrosine-kinase activity has been used in clinical trials as single or multiple agents and modalities with limited success (response rate 10–15%). The binding by ligands (EGF, TGF2, amphiregulin, and heparin binding – EGF) leads to antiphosphorylation of multiple tyrosine residues at the carboxy terminus, where SRC and other proteins interact with transducer mitogenic signals [39].

PI3k/AkT/mTOR Pathway Inhibitors Activation of these pathways plays an important role in the development and progression of HNSC. Mutation of the PI3k gene leads to cellular transformation of HNSC. Restoration of this pathway may lead to inhibition of PI3k phosphorylation and expression, which is responsible for radio resistance in HNSCC [50]. Also, activation of the AkT pathway may lead to EGFR overexpression and enhance resistance to targeted treatment. The mTOR has been shown to regulate critical cellular processes, including motility, proliferation, survival, and transcription. mTOR inhibition, however, may lead to negative feedback of the insulin-like growth factoral, which may lead to activation of PF3k and AkT and potentially counteracting the mTOR inhibitor [51]. Multiple agents or single agents targeting multiple pathways may be an ideal strategy. The complexity of the aberrant signaling in HNSCC underlines the difficulties in treating these patients (Fig. 3.2).

Structural Concept VEGF and FGF Elevated expression of VEGF and FGF and their receptors have been reported associated with angiogenesis and aggressive behavior in HNSC. The regulation of this growth factor is primarily through the hypoxia-inducible factor-1a (HIF1a)-dependent and -independent processes and involves both PI3k and AkT pathways [42–47]. A humanized VEGF monoclonal antibody (Bevacizumab) has recently been tested and shown to inhibit angiogenesis [48, 49].

Mesenchymal Epithelial Transformation In the last two decades, minimal attention has been paid to the role of epithelial/stromal interactions of invasion, progression, and metastasis in HNSC. Recent investigations in several solid tumor models have shown that invasion and metastasis are associated with alteration in cell to cell and cell to matrix adhesion altered epithelial cell polarity and increased motility. Several studies have shown that this process is initiated in response to extracellular stimuli

3  Cellular and Molecular Pathology of Head and Neck Tumors

and factors. Growth factors and their receptors play a central role in the transduction of key events associated with this process. Among the most important of these are the Ras, SRC, PI3k, and the MAP kinase pathways. The activation of these pathways have been shown to lead to downregulation of adhesion molecules (e.g., E-Cadherin) and elevation of surrogate mesenchymal markers (e.g., Vimentin) [3, 52, 53]. This process is highly relevant to squamous carcinoma invasion and metastasis, where E-Cadherin is a key adhesion molecule in squamous epithelial cells. E-Cadherin not only is important in cell to cell and cell basement adhesion, but also in mediating cell to cell cross-talk through Ca-dependent homotypic interactions [38, 54, 55]. Several growth factors, including TGFB, lead to downregulation of E-Cadherin and other cellular features associated with EMT. However, the manifestation of EMT in HNSC may vary considerably from tumor to tumor and within a given tumor. Not infrequently, minimal EMT changes are observed in well differentiated with broad invasive fronts while complete mesenchymal transformations is found in the sarcomatoid form of these tumors. In addition to the semiquantitative changes in these molecules, qualitative changes may also occur. This is clearly manifested in the phenotypic distribution of E-Cadherin from membranous to cytoplasmic localization. EMT, therefore, is a dynamic and heterogeneous process that underlies the biology of a squamous carcinoma and that the degree and extent of these changes reflect their aggressive nature.

Biomarker Applications in Head and Neck Tumorigenesis Early diagnosis in high risk individuals for HNSC is key to improving treatment and prognosis of this disease. Similarly, predicting the biological behavior, response to nonsurgical therapy and toxicity is important in stratifying patients for treatment and targeted therapy. Therefore, the identification of sensitive and reproducible markers is critical to the success of these efforts. The application of tissue-based assay requires that they accurately and reproducibly reflect the underlying pathological and biological processes. These processes are dynamically varied in and between individuals. Quantitation of lesional variabilities and confounding non-neoplastic processes is necessary for accurate interpretation and the exclusion of false positive and negative results. Integrating tissue assessment and biomarker results might ultimately be the best model of risk assessment for head and neck cancer patients [2, 24, 56].

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Salivary Gland Tumors Salivary gland tumors are rare and remarkably heterogeneous neoplasms of an uncertain histogenesis. They constitute only 2–3% of all head and neck neoplasms, with an overall incidence of approximately 2.5–3 per 100,000 ­persons per year [57, 58]. Major salivary glands are the most commonly afflicted sites, with 80% of tumors occurring in the parotid, 10–15% in the submandibular gland, and 5–10% in the sublingual and minor glands [59]. Most tumors (80%) of parotid gland origin are benign, whereas those arising in submandibular, sublingual, and minor glands are more often malignant. Primary malignant salivary gland neoplasms compose approximately 5–10% of all the head and neck carcinomas and 0.3% of all cancers [57]. Generally, salivary neoplasms present in middle and older age (mean age 56 years), with only 2–3% occurring in ­children under 10  years of age, and more commonly in males than in females [57, 60].

Salivary Tumors in Children The majority of salivary neoplasms in children are nonepithelial and mainly of vascular origin. The most common is mucoepidermoid followed by acinic cell carcinomas forming approximately 60% of malignant neoplasms in this category. The most common benign epithelial neoplasm in this age group is pleomorphic adenoma (PA). It is worth noting that a rare congenital tumor known as embryoma or sialoblastoma occur prenatally. Histologically, these tumors represent a neoplastic growth of embryonic, primitive, basaloid epithelial cell of salivary gland. These lesions are considered low-grade malignancy. The differential diagnosis is basal cell adenocarcinoma and adenoid cystic carcinoma (ACC) [61–63].

Fine Needle Aspiration in the Evaluation of Salivary Masses Fine needle aspiration (FNA) may be used in the initial evaluation of a salivary mass. The main indications of this procedure is to exclude lymphoreticular disorder, inflammatory and granulocytic reactive lesions and metastasis. FNA may not be recommended in the diagnosis of primary salivary gland tumors and cystic lesions. Not uncommonly, FNA may induce neurosis, reactive inflammatory, and reparative manifestations that may obscure the underlying neoplastic conditions. Occasionally, however,

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A.K. El-Naggar

especially in the planning of the extent of the operation, surgeons may utilize this technique to obtain a malignant diagnosis. Pathologic features of clinical importance: 1 . Tumor size 2. Histologic diagnosis 3. Malignancy grade (when applicable) 4. Margin status 5. Perineural involvement

Histopathology Table 3.1.

loss of heterozygosity at the long arm of chromosomes 8 and 12p loci [67, 70]. Two specific genetic markers have been consistently identified in PAs; the PLAG1 on chromosome 3p21 is the most frequent upregulated gene, but its biological significance in the development of pleomorphic adenoma remains uncertain [71]. The second recurrent and specific chromosomal alteration involving 12q14-15, leads to overexpression of the high mobility group A2 gene (HMGA2). The gene is an architectural factor that regulates transcription through binding to AT-rich DNA. Microarray analysis of PA and PLAG1transfected cells have identified most of the unregulated genes to be growth factors, such as IGF, BDGF1, CRABP2, SMARCD1, and EFNB1 [72]. Together these findings indicate that the PLAG1 gene contributes to oncogenesis through the induction of growth factors [73].

Warthin’s and Oncocytic Tumors

Benign Tumors Pleomorphic Adenomas PAs are the most common benign salivary tumors that primarily occur in the parotid. Clinically, these tumors pursue a benign clinical course with a tendency for local recurrence due to mainly nodular extension. Rarely, some PAs may metastasize while retaining their benign phenotypic features. Histologically they manifest varied cellular components, comprising epithelial and myoepithelial cells in variable background of myxoid and/or chondroid stroma [57, 64–66]. Karyotypic analyses have identified recurrent and specific cytogenetic abnormalities, with t(3;8) (p21;q12) reported in more than 40%, and a small subset manifesting rearrangements of the 12q14-15 region [67]. The latter include translocation involving 12q14-15 with chromosome 9p12 or different partners and/or inversion of both chromosomes at the same breakpoint. Random clonal abnormalities have also been detected in more than 20% of PAs [68, 69]. Molecular studies using microsatellite repeat markers reported frequent

Table 3.1  A simplified classification of salivary gland tumors

Myoepithelial/epithelial

Epithelial

Benign Myoepithelioma Pleomorphic adenoma

Oncocytoma Basal cell adenoma

Malignant (carcinoma) Myoepithelial Epi-myoepithelial Basaloid salivary Adenoid cystic Terminal duct

Mucoepidermoid Salivary duct Adenoid cystic, solid Basaloid salivary Acinic cell

Warthin’s tumor (WT) is the second most common benign salivary gland tumor. It arises almost exclusively in intra- or periparotid lymphoid stroma. Histopathologically, the tumor manifests oncotypic epithelial cell proliferation within lymphoid stroma with and without cystic formation. A spectrum of oncocytic tumors ranging from nodular oncocytic hyperplasia, adenoma, and carcinoma have been described and most likely related to Warthin’s tumors [74]. Current molecular and cytogenetic studies indicate that the majority of these lesions manifest a normal karyotype [75], while approximately 10% have cytogenetic abnormalities; the most common cytogenetic alteration identified is the t(11;19) (q21-22;p13) [76, 77]. The same translocation and its fusion gene product CRTC1/MAML2 were also found in mucoepidermoid carcinoma (MEC). The finding of this abnormality in both tumors, along with their reported simultaneous occurrence, indicates a genetic link between these lesions. Collectively, the data support a clonal origin in a subset of these tumors with a propensity to transformation to MEC or oncocytic carcinoma.

Basal Cell Tumors Both basal cell adenomas and carcinomas are rare and constitute approximately 2–3% of all salivary gland tumors. These tumors may not infrequently pose diagnostic difficulties due to their cytomorphologic similarities. They are typically formed of bland basal cell proliferation in nests and/or cords formation with intercellular eosinophilic homogenous material deposition [78]. Because of the infrequency of these tumors, only small numbers have been genetically analyzed; a common cytogenetic alteration in few tumors was a trisomy

3  Cellular and Molecular Pathology of Head and Neck Tumors

8, but other sporadic cytogenetic alterations, including t(7;13) translocation, have also been reported [79]. CGH analyses of examples of these tumors showed loss of chromosomes 2, 6, and 7, gains of chromosomes 1 and 8, and amplification of 12q region. Molecular analysis of these tumors has reported frequent loss of heterozygosity at chromosome 16q12-13, a region that houses the cylindromatosis gene (CYLD) [79].

Canalicular Adenoma Canalicular adenoma is characterized by columnar epithelial cells forming anastomosing bilayered cellular formations including nests and is trabecular in a vascular stroma. The lesions are typically well circumscribed and encapsulated [57, 65]. Differential diagnosis of canalicular adenoma from basal cell adenoma and ACC may occasionally be difficult, especially on biopsy specimens. Because of their rarity and benign nature, molecular studies of this entity are very rare. Myoepithelial Tumor Myoepithelial tumors are formed almost exclusively of myoepithelial cells, which are rare and are less than 1% of all salivary gland neoplasms. Some tumors may show focal areas of pleomorphic adenoma. They may manifest a variety of phenotypic forms, including plasmacytoid, spindle, clear, and/or epithelial features. Current molecular genetic data on these lesions are sparse and preclude any definitive findings that contribute to either their development or biology. Cytogenetic analyses of a few examples have reported nonspecific chromosomal abnormalities and were insufficient for comment on their contribution to these tumors [80, 81]. Upregulation of the WT1 mRNA has been detected in some benign and malignant myoepithelial tumors, but the oncogenic role of this event in their development is unknown [82].

Fig. 3.3  Ductal structure and proposed origin of salivary gland tumors

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Malignant Tumors Mucoepidermoid Carcinoma MECs compose approximately 30% of malignant salivary neoplasms and are most common in children and adolescents. MEC manifests three distinctive phenotypic grades based on the cellularity and architectural features of the tumors. Of all salivary neoplasms, MEC is the only entity in which both cytogenetic and molecular analyses have led to the identification of consistent unique alteration that may constitute an initiating event in the development of a subset of these tumors. Several cytogenetic analyses of MEC have shown translocation t(11;19) (q21;p13) either alone or with other nonspecific alterations [75, 83–85]. Cloning of this translocation has identified a fusion oncogene composed of exon 1 of the MECT1 (CRTC1/WAMTP) gene (Fig. 3.3) on chromosome 19p13 and exons 2–5 of the MAML2 gene on chromosome 11q21 regions [86]. MAML2, a member of the mastermind gene family, encodes a nuclear protein that binds to the CSL transcriptional factor and the intracellular domain of the Notch receptor to activate the Notch target gene. The fusion partner is the CRTC1 (MECT1), a member of the highly conserved CREb/cAMP coactivator gene family [87, 88]. Studies of this fusion transcript in a series of MEC have reported a correlation between fusionpositive tumors and low tumor grade and better behavior. Fusion-negative MEC may evolve from a different evolutionary pathway and may represent a biologically distinctive category. The results also suggest that tumors lacking the fusion transcript behave more aggressively. The finding of the fusion transcript in both sporadic Warthin’s tumor and MEC and concomitant tumors supports an early or etiologic role in the development of a subset of these tumors. Epithelial ductal cells in heterotypic salivary tissue in intra- or paraparotid lymphoid stroma acquiring the t(11;19) fusion gene give rise to Warthin’s tumor, while the same alteration in the salivary tissue gives rise to MEC in sporadic presentations.

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The development of MEC in a Warthin’s tumor may therefore result from metaplastic changes in ductal cells with the fusion transcript [89–92].

Salivary Duct Carcinoma and Adenocarcinoma Ex-Pleomorphic Adenoma Salivary duct carcinoma (SDC) and adenocarcinomas present either de novo or in the setting of pleomorphic adenoma and manifest remarkable similarity to mammary duct carcinoma [93, 94]. Cytogenetic studies of some of these tumors have shown rearrangements of chromosome 8q12, alteration of chromosome 12q13-15 region, and amplification of both the HMG1C and MDM2 genes may be potentially associated with these tumors. Other studies have shown that translocations of chromosome 5(q22-23, q32-33) and t(10;12) (p15;q14-15) resulted in transportations of the entire HMG1C gene to chromosome 10 marker [57, 95–97]. Using microsatellite markers on microdissected benign and matching malignant components of salivary gland carcinoma ex-pleomorphic adenoma (Ca ExPA), have shown alterations at 8q and/or 12q in both components and restricted alterations at chromosome 17p loci in the malignant component [95, 96]. These findings suggest that alterations at 8q and 12q regions represent early events, whereas alteration at 17p is associated or coincident with the malignant transformation. Studies of specific genes and loci have also reported homozygous deletion of the p16 gene on chromosome 9p21 [98, 99], and p53 alterations and loss of heterozygosity at different loci on chromosome [73]. A subset of SDC, as in mammary ductal carcinoma express hormonal and growth factor overexpression that may be used in their biological and therapeutic stratification [98]. Overexpression of HER2, EGFR, and androgen receptors are found in more than one-third of these tumors [100, 101].

Adenoid Cystic Carcinoma ACC is the second most common malignant salivary gland tumor and the most clinically relentless malignancy. ACC is known for its indolent and persistent clinical behavior and propensity for perineural invasion. ACC manifests three phenotypic subtypes, which they nearly always present in the majority of tumors but with variable proportions [102]. These include tubular, cribriform, and the solid morphologic variants. In both the tubular and the cribriform phenotypes, the tumor units consist of myoepithelial and ductal epithelial cells. Cytogenetic studies of these tumors have reported frequent alterations at chromosomes 6p, 9p, and 17p, with the most consistent alteration at the 6q regions (Table 3.2) [99, 103].

A.K. El-Naggar Table 3.2  Adenoid cystic carcinoma (ACC)

Acinic cell carcinoma

Pleomorphic adenoma Adenoid cystic carcinoma Monomorphic adenomas Epithelial myoepithelial carcinomas

Warthin’s Oncocytoma

Mucoepidermoid carcinoma Adenocarcinoma

Studies of ACC found a high frequency of loss of heterozygosity at 6q23-25, and this correlated with histologic grade and clinical behavior. Studies using microsatellite markers have also reported frequent loss at chromosomes 12q, 6q23qter, 13q21-33, and 19q regions. These regions house two genes, PLAGL1 and LATS, that were not mutated in any of these tumors. A recent comparative genomic hybridization of ACCs identified a novel gain at chromosome 22q13 region in 30% of the tumors in addition to the loss of chromosome 6q and gains of chromosomes 16p and 17q regions [104– 106]. Microarray analysis of a few examples of these tumors have shown amplification of MDM2, HMG1C, MYC, and other genes located on chromosomes 8q and 12q14 [107– 109]. A frequent finding in these tumors is the overexpression of the C-Kit protein. C-Kit (CD117) is a transmembrane tyrosine kinase receptor encoded by the C-Kit gene on chromosome 4. The C-Kit ligand, a stem cell factor (also known as steel factor and mast cell growth factor) induces signal transduction pathways affecting development, cell growth, and migration of different cell functions [110–112]. The role and the cellular distribution of this gene product in the biology and as a target in these tumors remain to be determined.

Acinic Cell Carcinomas Acinic cell carcinoma is a distinctive salivary malignancy that develops almost exclusively in the parotid gland. These tumors arise from acinar cells and manifest granular serous cellular features with variable and overlapping morphologic subtypes [113]. They are generally low-grade indolent carcinomas, occasionally presenting as high-grade carcinomas with high mitotic figures, necrosis, and lymph node metastasis [114]. In addition, several examples of transformation into dedifferentiation or anaplastic carcinomas have been reported. Cytogenetic and molecular studies of these tumors are few and inconclusive. One study cites evidence for a frequent loss of heterozygosity at limited chromosomal regions [115], including 4p15-16, 6p25-qter, and 17p11, suggesting that these regions may contain critical genes related to their development. In another study of multiple samples of an ACC, variable clonal alterations were obtained, suggesting

3  Cellular and Molecular Pathology of Head and Neck Tumors

multiclonal origin [116]. Studies of dedifferentiated acinic cell carcinoma have shown an association of such transformation with Cyclin D1 upregulation. The lack of confirmatory and validation follow-up studies precludes any speculation on the role of these findings in this entity.

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type. They may arise in either intraparotid lymph nodes or the parenchyma. The vast majority are of the follicular B-cell derivation with rare instances of T-cell origin.

Metastasis to Salivary Glands Polymorphous Salivary Adenocarcinoma (Terminal Duct Carcinoma) This entity is characterized by intratumoral growth pattern variabilities and uniformed monotonous cellular composition. The hard palate is the most frequent site but they may rarely occur in major salivary glands. The tumor constitutes 19.6% of malignant minor gland tumors. Because of the lack of encapsulations, these tumors typically infiltrate adjacent tissue and are prone to perineural invasions. The recurrence rate for these tumors is approximately 17% and regional metastasis occurs in approximately 9% [117].

The most common metastasis to major salivary glands, especially the parotid, is squamous carcinoma followed by melanoma of the skin. This is largely due to the lymphatic drainage of skin of the face. Hematogenous spread to the parotid originates primarily from kidney, breast, and lung carcinomas. Metastasis to the submandibular gland is very rare due to the lack of intraglandular lymph nodes. Epithelial neoplasms are rarer and disproportionately malignant [57].

Genomic and Proteomics of Salivary Gland Tumors

Epi-Myoepithelial Carcinoma This rare entity represents a malignancy of low grade and indolent course that is composed of dual myoepithelial and ductal tumor cells. Histopathologically, the tumor forms duct and tubular formations of relatively prominent clear myoepithelial cells and inner cuboidal and uniform duct cells. Rare Salivary Gland Neoplasms and Subjects Squamous Carcinoma Rarely squamous carcinoma may arise de novo in major salivary glands and if presented not underlined. The exclusion of metastasis from other sites must be proved. Rare carcinomas reported to be of primary origin include small cell and lymphoepithelial carcinoma. Nonepithelial Neoplasms Nonepithelial neoplasms form less than 5% of all salivary gland tumors. They represent lesions arising from salivary gland supporting connective tissue. The most common lesions are angioma, lipoma, neurofibroma, and hemangiopericytoma. The growth and microscopic features of these lesions are identical to those encountered in other sites. Primary Lymphoma Lymphomas are very rare and mainly found in the parotid gland. The majority of primary lymphomas are of the MALT

Proteomic analysis of solid tumors remains limited and difficult to execute. There is only a single study of ACC xenografts by fluorescent two-dimensional gel, electrophoresis, and matrix-assisted laser-desorption/ionization techniques. This study identified four upregulated and five downregulated proteins. Of these proteins, maspin and stathmin were confirmed to be highly expressed in human ACC. Similar attempts have been made in some salivary gland tumors. The results, however, should be considered preliminary or suspect until verified [72, 93, 118, 119].

Thyroid and Parathyroid Tumors Thyroid Thyroid nodules are one of the most common clinical conditions. The vast majority of these are reactive lesions or benign tumors and only 10% are malignant. Approximately 14,000 new cases of thyroid carcinomas are diagnosed per year in the USA [120]. The histologic subtypes of thyroid malignancies include papillary, follicular, poorly differentiated, anaplastic, and medullary carcinomas. Broadly, these tumors can be categorized into differentiated (papillary, follicular, and medullary) and undifferentiated (poorly differentiated and anaplastic) carcinomas [121, 122]. The papillary, follicular, poorly differentiated (insular), and the majority of anaplastic carcinomas arise from the follicular epithelial cells while the MTC is derived from parafollicular calcitonin-­ producing c-cells [123–126].

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Etiology The etiology of thyroid malignancies is largely unknown, although exposure to radiation during childhood (papillary) and iodine deficiency (follicular) have been linked to the development of certain carcinoma subtypes. Papillary thyroid carcinoma may affect any age, but especially children, young adults, and females. Carcinomas typically present as an enlarged mass with or without ipsilateral nodal involvement [127–130]. Initial radioscintigraphy is helpful in distinguishing between hot (benign) and cold (malignant) nodules [131]. Pathology Cytology Fine needle aspiration (FNA) is the first line of diagnostic techniques for thyroid tumor diagnosis. In general, an accurate diagnosis of papillary and medullary thyroid carcinoma can be readily made on FNA. The sensitivity and the specificity of FNA in diagnosing follicular lesions, including follicular variant of papillary carcinoma, however, is low. It is estimated that up to 30% of FNA-based diagnosis of follicular neoplasms are indeterminate [132, 133]. Histology Thyroid neoplasms are generally classified based on their histogenesis from epithelial (follicular cell) and neuroectodermal (C-cell) neoplasms. Epithelial neoplasms are broadly benign follicular adenomas and differentiated neoplasms and poorly differentiated and anaplastic carcinomas. Follicular Adenoma Adenomas are characterized by a well-circumscribe nodular growth with thin encapsulation. They may present as solitary or multiple nodules at any age and gender. Microscopically, they may manifest microfollicular, trabecular, and macrofollicular forms. The main differential diagnoses for adenomas are follicular hyperplasia (Goiter) and follicular carcinoma. Oncocytic changes due to the high content of mitochondria are most likely secondary to respiratory cellular demands. The biological behavior of these neoplasms is similar to those of corresponding follicular tumors [134–136]. Differentiated Carcinomas • Follicular type Follicular carcinomas comprise approximately 5–10% of all thyroid malignancies. They generally afflict females in

A.K. El-Naggar

their middle age than males. A high incidence of these tumors is reported in iodine deficient regions, suggesting a role for continuous TSH stimulation in the genesis of this entity. The diagnosis of this entity is based on the findings of a thick fibrous capsule and the presence of capsular and/or vascular penetration [134]. These tumors can be further classified as minimally invasive or encapsulated, if invasion did not extend beyond the capsule. Follicular carcinoma is typically solitary and may present or be preceded by metastasis typical to bone, lung, and brain [125, 137–139]. Patients present with a single palpable cold mass with a high propensity for radioactive iodine uptake [140, 141]. • Papillary type Papillary carcinoma is the most common of all thyroid carcinomas, accounting for more than 70% of these tumors. They may present at any age with peak incidence between 30 and 40 years of age. Females are far more affected than males, and young patients typically have a better and long protracted course than older patients, especially men. There is strong circumstantial evidence linking Hashimoto’s thyroiditis to increased incidence of papillary thyroid carcinoma [142–144]. Papillary thyroid carcinomas are multifocal in more than 75% of the cases and total thryoidectomy is generally the treatment of choice. Papillary thyroid carcinoma may present as a thyroid mass (80%) or as a lymph node metastasis (20%). The hallmark of papillary carcinoma is finding papillary structures lined by cuboidal or columnar cells with clear and/or cleaved nuclei. The nuclear features are especially helpful in the diagnosis of the follicular variant of this entity. Not uncommonly present (40%) is the concentric calcification associated with this tumor (psammoma bodies). Several histopathologic variants of this entity have been described with some being associated with a more aggressive clinical course. However, the lack of prospective studies with long-term follow-up render the significance of these subtypes tenuous. The clinical aggressiveness of papillary thyroid carcinoma varies depending on the gender, age, and size of the tumor with older males having a more aggressive course as well as patients with large invasive tumors [120, 126]. • Undifferentiated carcinomas (a) Poorly differentiated This histologic variant represents a tumor that lacks follicular or papillary differentiation and the cellular anaplasia of anaplastic carcinoma. Tumors typically manifest cell nests or cords with monotonous cellular features. The differential diagnosis is mostly with medullary thyroid carcinoma. Tumor cells react positively to antithyroglobulin antibodies and they are negative for calcitonin. Their behavior is considered more aggressive than the fully differentiated tumors [122, 145].

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(b) Anaplastic Anaplastic thyroid carcinoma (ATC) is the most clinically aggressive neoplasm and accounts for 4–10% of all thyroid malignancies. This entity afflicts elderly individuals and is more common in females than males (3:1) [146, 147]. Clinically, patients present with rapidly progressive local disease. The majority of these tumors arise from preexisting differentiated thyroid carcinoma, most commonly the papillary phenotype. In resected specimens of these tumors, evidence for a differentiated carcinoma can be found. The etiology of ATC is unknown, but previous radiation of thyroid lesions has been linked to the development of these tumors. Histopathologically, these tumors manifest highly malignant tumor cell composition with heterogeneous features and tumor necrosis. The most common pathologic phenotypes are sarcomatous, giant cell, and squamous variants. The differential diagnoses of these tumors include sarcomatoid carcinoma of the upper aerodigestive tract, sarcoma, and melanoma [121, 148–150]. Immunostaining assists in excluding sarcoma and melanoma. The prognosis of these patients is very poor. • Medullary carcinoma Medullary thyroid carcinoma arises from the C-cell, a neuroectodermally derived cell, and accounts for 3–10% of thyroid cancer. The tumors present in two forms: sporadic, the most common, which accounts for 70–80% and the familial form, which represents the remaining 20–30%. The tumors affect both genders equally and patients in middle age. The familial and the sporadic forms have mutation in the RET gene, the frequency and the type of these mutations vary. Tumors in the sporadic form present with a solitary mass with/or without neck enlargement and paraneoplastic syndrome. Tumors in the familial form are generally multifocal and affect the younger age and children [151, 152]. The most common location of these tumors is the lateral aspect of the upper 2/3 of the thyroid lobes, where a high aggregation of C-cells can be found. Histopathologically, tumors consist of nests and cords and organized structures composed of small to medium-sized cells with uniform nuclei. Tumor clusters are encircled by delicate vessels and fibrous tissue. Not uncommonly, deposition of dense homogenous eosinophotic materials representing amyloid deposition is noted. The amyloid nature of these materials can be verified by either congo red staining or by light microscopic birefringence [52]. Immunostaining for calcitonin and other neuroendocrine markers may be used for confirmation. The most common sites of metastasis for MTC are regional lymph nodes, lung, liver, and bone. The prognosis of MTC depends on several factors, including age, gender, size,

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and stage. Generally, the young and females have better outcomes. Patients with MEN-2B have a worse outcome. The differential diagnosis of these tumors include metastasis from neuroendocrine carcinoma, renal cell carcinoma, and microfollicular thyroid neoplasm. • Sclerosing mucoepidermoid carcinoma This is a rare malignancy of the thyroid gland, typically in association with Hashimoto’s thyroiditis. It is characterized by infiltrating sclerotic stroma with infiltrating nests of squamoid cells with occasional mucinous cells. The stroma is characteristically infiltrated by numerous eosinophilis.

Molecular Analysis of Thyroid Neoplasms Genetics RAS gene mutations were frequently found not only in thyroid carcinomas but also in adenoma [153]. Point mutations in RAS have been linked to early thyroid tumorigenesis. Whether adenomas with RAS gene mutations represent a biologically malignant lesion remains unknown [154–157]. Rearrangements of the PPARg/RAX8 translocation have also been reported in follicular carcinoma and adenomas suggesting that it may constitute an early event in their development [158–162]. Several studies have also shown mutation in the RAS gene RET/PTC rearrangements on chromosome 10 and BRAF oncogene mutations in thyroid carcinoma. The frequency and the biological significance of these events are the subject of debate and remain to be determined. The most frequent of these genetic alterations is the BRAF point mutation in Exon 15 at codon 600 [129, 163–166]. This mutation has been reported in up to 70% of PTC cases. RET mutated MTC are characterized by early onset and metastasis to lymph nodes and distant organs [152, 167]. The RET proto-oncogene encodes a receptor tyrosine kinase (RTR) that is widely expressed in neuroendocrine cells. RET point mutation in the intracellular kinase domain or extracellular occur in medullary thyroid carcinoma [171, 172]. RET gene rearrangements; however, are associated with papillary thyroid carcinoma. The common underlying denominator in tumor growth is the constitutive activation of the RET kinase [143, 168–170]. The molecular mechanisms that result in RET activation and the pathophysiology vary widely [87]. PTC with RET gene arrangements are heterogeneous and generally indolent and rarely present with metastasis. In these tumors, chromosomal rearrangements involving the RET gene fuse the 5¢ end and a promoter of a gene upstream of the RET kinase domain leading to the expression of a chimeric product, a RET/PTC. RET/PTCs are localized to the

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cytoplasm since they lack the NH2 terminal sequence and the transmembrane domain of the RET gene. All NH2-terminal fusion partners identified to date contain homodimerization domains that mediate dimerization and activation of the kinase region in RET/PTC oncoproteins [168, 169, 172–174]. Recent studies have also established the anaplastic phenotypic transformation from differentiated thyroid carcinoma through the analysis of RAS, BRAF genes [146, 147, 156, 175–177]. Galectin-3 is an antiapoptic molecule of the B-galactoside binding lectin family. Alteration in the expression of galactin-3 has been proposed as a diagnostic marker of thyroid malignancy [131, 133, 178, 179]. Genomics Gene expression analysis of several thyroid neoplasms have been performed. Upregulation of MET, SGRPINA, FNI, CD44, and DPP4 and downregulation of TFF3 gene have been reported in some of these studies [160, 178, 180–183]. Genomic analysis although allowed for the identification of thyroid neoplasm and the biological categorization within carcinomas, the utilization of these assays in the clinical diagnosis is limited and impractical.

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Parathyroid Adenoma Parathyroid adenoma is a benign parathyroid gland neoplasm and is the most common cause of hyperparathyroidism accounting for more than 80% of cases. Parathyroid adenoma affects more females than males in the middle age. These lesions are considered clonal in origin and present as a single well-circumscribed nodule with a peripheral rim of parathyroid tissue [127, 189]. Adenoma is typically homogenous and contains no adipose tissue cells. Although, they may arise in any gland, they are more frequently reported in the lower glands [120].

Locally Infiltrative Parathyroid Neoplasm (Atypical Parathyroid Adenoma) Occasionally, parathyroid neoplasms with cytomorphologic features identical to those of hyperplasia or adenoma and infiltrative growth into surrounding soft tissue with intersecting fibrous bands may be encountered. The lack of high and abnormal mitotic figures, necrosis and marked cellular pleomorphism preclude a definitive malignant diagnosis. These lesions are typically prone to local recurrence because of the difficulties to completely excise them. These lesions may also be called atypical parathyroid adenoma.

Parathyroid Lesions Parathyroid glands are derived from the third and fourth pharyngeal pouches and are recognized by the fifth to the 6 weeks of gestation. The majority of humans have two pairs of parathyroid glands. Multiples up to 10 (13%), and as few as one, have been reported in humans. Normal glands are encapsulated, small, soft, and tan to red-brown in color. Parathyroid cells are organized in lobules with fat cells and vascular stroma. The degree of fat in normal parathyroid varies but in general is approximately 60%. Although, literally a non-neoplastic process, evidence of clonality and evolution to adenoma and carcinoma based on clonality analysis has been documented [184–187].

Parathyroid Carcinoma Parathyroid carcinoma is a rare, highly malignant neoplasm accounting for less than 5% of patients with hyperparathyroidism. This entity may be hormonally active or inactive [190]. The inactive carcinoma has reportedly been more aggressive. These tumors present as a solid mass that are difficult to excise due to its infiltrative nature. Histopathologically, these tumors are characterized by a proliferation of markedly pleomorphic cells, high and abnormally mitotic figures, broad intersecting fibrous bands, vascular and soft tissue invasion and necrosis. This is a surgically treated disease but more than a third of these patients experience metastasis.

Parathyroid Hyperplasia Molecular Analysis of Parathyroid Lesions Parathyroid hyperplasia is pathologically characterized by increased parathyroid cells with reduction of fat cells in parathyroid lobules. This may occur in all four glands with a variable degree. Generally, this may signify a systemic etiology such as calcium deficiency, vitamin D alterations, or kidney diseases. Hyperplasia of the parathyroid can also be a manifestation of MEN type I syndrome. Histopathologically, they manifest diffuse or nodular cellular proliferation. The cellular feature varies and may include clear and oncocytic cytoplasm [188].

Alterations in overexpression of Cyclin D and chromosome 11q13 regions have been shown to characterize parathyroid nodular hyperplasia and adenoma. Other clonal and molecular findings support a clonal basis for the development of at least a subset of these lesions. The Cyclin D and retinoblastoma glue have frequently been found in parathyroid carcinoma alterations [120, 191–193]. Mutation at the MEN1 gene on chromosome 11q13 region has been reported in up

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to 50%. Genome-wide studies have also shown loss of 11q region in addition to other chromosomes [194–196]. Molecular alterations of parathyroid carcinoma are rare and inconclusive, but alterations of the retinoblastoma and the MEN1 genes have been reported. Proteins have reported to be limited to these tumors. Loss of heterozygosity and mutation of the HRPT2 gene, which encodes for the parafibromin has also been documented in parathyroid carcinoma and are believed to be restricted to malignancy. If validated, may have a diagnostic and therapeutic implication [197–199]. Somatic mutations as well as germline mutations of the HPRT2 have been implicated to underlie primary hyperparathyroidism [200].

Sinonasal and Skull Base Tumors A wide spectrum of malignant neoplasms arises from the sinonasal and skull base regions. The majority of these tumors are poorly or undifferentiated malignancies and manifests overlapping features resulting in diagnostic challenges [201, 202]. Excluding tumor-like lesions like hamartomas and teratomas, the most commonly encountered benign neoplasms are Schneiderian papillomas.

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squamous carcinoma with and without evidence of dysplasia. The presence of keratinization is always associated with carcinoma. The differential diagnosis of inverted papilloma includes other forms of Schneiderian papilloma. Recurrence rate is approximately 45–75%. Molecular studies of these lesions are rare. However, evidence for monoclonity has been reported, but no specific genetic alterations were linked to progression [203].

Salivary Type Neoplasms Salivary tumors arising at these locations derived from minor glands and manifest identical morphologic features to those arising in major and minor salivary glands. The difference is their uncapsulated nature and the associated difficulties in assessing margin status. The most common benign tumor is pleomorphic adenoma and the most common malignancies are adenoid cystic, mucoepidermoid and acinic cell carcinomas in descending order and adenocarcinoma, not otherwise classified. The differential diagnosis is mainly from metastasis and nonsalivary seromucinous carcinoma [202].

Nonsalivary Type Adenocarcinoma Schneiderian Papillomas Schneiderian papillomas account for 0.4–5.0% of all sinonasal tumors and are classified based on their growth and histological features into exophytic, inverted and cylindrical subtypes. The exophytic form arises predominantly in the nasal septum, but they may also occur in the nasal cavity and the maxillary sinus. They are usually solitary and rarely associated with malignant transformations. Histologically, they manifest a fibrovascular core lined by hyperplastic nonkeratinizing squamous and/or transitional epithelium. The main differential diagnosis is papillary squamous carcinoma. The latter exhibits cellular features of malignancy and stromal invasion. These lesions are prone to recurrence in up to 22–40% of the cases. Inverted papillomas comprise approximately 45% of all papillomas and are characterized by inward growth due to invagination of the epithelial components into the stroma. They commonly arise in the nasal cavity and paranasal sinuses and rarely in the septum. These lesions are also known for high recurrence rate and progression into carcinoma [203]. The epithelial lining of the inverted papilloma is commonly nonkeratinizing, stratified, squamous epithelium with vacuolation, intraepithelial microcysts, and acute inflammatory cells. Malignant transformation may present as differentiated or poorly differentiated

These adenocarcinomas are classified into seromucinous type and intestinal type. The seromucinous type most likely arise from the seromucinous glands lining, the respiratory epithelium of the nasal cavity. They are typically welldifferentiated adenocarcinoma. The intestinal type is similar to adenocarcinoma of the colorectal sites. These tumors arise from the respiratory epithelium most likely due to intestinal metaplasia as a result of exposure to wood dust or leather chemical processing. These tumors affect middle and elderly individuals with the aforementioned risk factors. The tumors manifest identical phenotypic features to their intestinal counterparts, including mucinous production and signetring formation. The biological behavior of these tumors is generally aggressive with the majority of patients succumbing to their disease within 3 years. Molecular and phenotypic studies of this entity have shown evidence for shared molecular alterations with colonic adenocarcinoma [204–208].

Squamous Carcinoma Carcinomas of the sinonasal cavity comprise approximately 3% of all malignant tumors. The majority (70%) are squamous in derivation. The vast majority occurs in the maxillary sinus

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and a small subset occurs in other nasal sites. Several etiologic factors have been linked to the development of these tumors, among which nickel and thorotrast exposure were the most commonly incriminated. These tumors typically affect men in their 50s to 60s. Histopathologically, they may present as keratinizing squamous carcinoma or nonkeratinizing [201, 209]. Other forms of squamous carcinomas as verrucous and spindle cell and basaloid squamous carcinomas have been described. The differential diagnoses of these tumors include metastasis, ameloblastomas, and inverted papilloma. The biological behaviors of this entity depend on the site and degree of differentiation with the nasal carcinoma patients fairing better than those with paranasal tumors [202].

Undifferentiated Sinonasal Carcinoma These tumors are characterized by their lack of differentiation and affects both males and females equally. Histologically, they manifest undifferentiated carcinoma similar to those of type III NPC. These tumors run an aggressive biological course and present in advanced stage. Because of the undifferentiated nature, they may be confused with a wide variety of undifferentiated neoplasms at these sites. These include poorly differentiated squamous carcinoma, NPC, neuroblastoma, melanoma, lymphoma, and small round cell tumors. Immunohistochemical and molecular markers are important in differentiating these tumors, especially on small pretreatment biopsies [202, 210, 211].

Neuroendocrine Carcinomas Neuroendocrine carcinomas of the sinonasal region are uncommon relative to the larynx and are classified into typical (well-differentiated) and atypical carcinoid (moderately differentiated) and poorly differentiated (small and large cell) carcinoma. The most common subtype is the poorly differentiated subtype, which typically affects the nasal cavity with extension to the ethmoid and maxillary sinuses. They affect men and women equally with a wide range of age. The diagnosis and differential diagnosis is established by performing keratin and other neuroendocrine markers [212–214].

Small Round Cell Tumors and Neuroblastoma A host of tumors that share a small rounded and basal-like tumor cell composition is not uncommonly presented at these sites. These include neuroblastoma, rhabdomyosarcoma, neuroendocrine carcinoma (small cell), and Ewing’s/

A.K. El-Naggar

neuroectodermal tumors [215–217]. Although younger age groups are more frequently affected, older ages may also be presented with these tumors. They occur equally in both sexes. There are no known predisposing factors associated with the development of these tumors and most likely familial and genetic factors may underlie their development. The diagnosis of these tumors, especially on initial biopsy, is challenging and is largely aided by ancillary immunohistochemical and molecular markers [201, 217–221]. Sinonasal Melanoma Primary sinonasal melanoma is very rare and accounts for 1% of all melanomas and 2.4% of nasal malignancies. The most common sites for this entity is nasal cavity and the paranasal sinuses with the most frequent sites being the nasal septum, lateral nasal wall, and the middle and inferior turbinates. Histologically, cells are small, rounded and undifferentiated and commonly manifest melanin pigment. These tumors are highly aggressive and prone to recurrence. They are typically presented at middle or older age, but they may present at any age. The differential diagnosis of this tumor includes all small round undifferentiated tumors at these locations (Fig. 3.4) [216, 222–225]. Fibrous and Vascular Neoplasms These tumors are divided into a benign, low-grade category and include fibromatosis, fibroma, myxoma, hemangioma, Schwannoma and hemangiopericytoma and solitary fibrous tumor and low-grade fibrosarcoma. Their diagnosis is based on the histopathologic features and their treatment is largely surgical [202]. Odontogenic Tumors Odontogenic lesions may also present in the sinonasal sites especially the maxillary sinus and includes calcifying odontogenic and tumor ameloblastoma. The most important differential diagnosis for these tumors is inverted squamous papilloma and squamous carcinoma. These tumors typically occur in young and middle age individuals and behave as benign or locally destructive tumors. Ameloblastoma may however, transform into more malignant ameloblastic carcinoma. Complete excision of these tumors is curative. Teratocarcinosarcoma Teratocarcinosarcoma is an extremely rare carcinoma that may lead to management difficulties. The histogenesis of

3  Cellular and Molecular Pathology of Head and Neck Tumors

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Fig. 3.4  Algorithmic marker applications for sinonasal undifferentiated neoplasms

this entity remains unsettled, but an origin from stem cell is possible. Histologically, these tumors are characterized by the presence of immature neural elements and malignant epithelial and mesenchymal tumors. The tumor affects mainly men in their middle and old age. These tumors are treated surgically with postoperative radiotherapy [226].

Lymphoproliferative Disorders Non-Hodgkin lymphoma is the most common lymphoproliferative disease in the sinonasal tract. Of the different subtypes that represent this category, the Nk1 T-cell lymphoma is the dominant lymphoma at these sites. T-cell lymphoma (natural killer) typically afflicts predominantly men in the middle or old age. The disease has been reported to be more common in Asians. The most common presentation is destructive mid-facial lesions with obstructive symptoms. The disease is strongly associated with EBV. Histologically, the disease is characterized by polymorphous cell infiltrate, including lymphocytes, plasma cells, histiocytes, and eosinophils with necrosis [227–231]. The differential diagnosis of this entity includes infectious conditions, especially fungal organisms, and especially Wegener’s granulomatosis. The absence of EBV virus and antineutrophil cytoplasmic antibodies exclude the latter.

Molecular and Genetic Advances in molecular genetic studies of skull-base neoplasms are limited to small round cell tumors, including Ewing’s, synovial, and rhabdomyosarcomas. Specific trans-

location generating oncogenic fusion transcripts have been identified in some of these tumors and currently used in their diagnosis and management stratification. In Ewing’s sarcoma and peripheral primitive neuroectodermal tumor, the EWS/FLI-1 gene resulting from the t(11;22) (q24;q12) is detected in 80% of tumors. The fusion gene has also been detected in neuroblastoma and rhabdomyosarcoma [220, 221, 232]. The PAX-FKHR fusion gene has also been used in the diagnosis and to guide treatments in alveolar rhabdomyosarcoma. Future identification of specific translocation will lead to better diagnosis and classification of other tumors.

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3  Cellular and Molecular Pathology of Head and Neck Tumors 214. Milroy CM, Ferlito A. Immunohistochemical markers in the diagnosis of neuroendocrine neoplasms of the head and neck. Ann Otol Rhinol Laryngol. 1995;104:413–8. 215. Bourne TD, Bellizzi AM, Stelow EB, et  al. p63 Expression in olfactory neuroblastoma and other small cell tumors of the sinonasal tract. Am J Clin Pathol. 2008;130:213–8. 216. Fujimura Y, Ohno T, Siddique H, et  al. The EWS-ATF-1 gene involved in malignant melanoma of soft parts with t(12;22) chromosome translocation, encodes a constitutive transcriptional activator. Oncogene. 1996;12:159–67. 217. Kumar S, Perlman E, Pack S, et al. Absence of EWS/FLI1 fusion in olfactory neuroblastomas indicates these tumors do not belong to the Ewing’s sarcoma family. Hum Pathol. 1999;30:1356–60. 218. Babin E, Rouleau V, Vedrine PO, et al. Small cell neuroendocrine carcinoma of the nasal cavity and paranasal sinuses. J Laryngol Otol. 2006;120:289–97. 219. Brissett AE, Olsen KD, Kasperbauer JL, et al. Merkel cell carcinoma of the head and neck: a retrospective case series. Head Neck. 2002;24:982–8. 220. Gardner LJ, Ayala AG, Monforte HL, et al. Ewing sarcoma/peripheral primitive neuroectodermal tumor: adult abdominal tumors with an Ewing sarcoma gene rearrangement demonstrated by fluorescence in situ hybridization in paraffin sections. Appl Immunohistochem Mol Morphol. 2004;12:160–5. 221. Qian X, Jin L, Shearer BM, et al. Molecular diagnosis of Ewing’s sarcoma/primitive neuroectodermal tumor in formalin-fixed paraffin-embedded tissues by RT-PCR and fluorescence in situ hybridization. Diagn Mol Pathol. 2005;14:23–8. 222. Chang AE, Karnell LH, Menck HR. The National Cancer Data Base report on cutaneous and noncutaneous melanoma: a summary of 84,836 cases from the past decade. The American College

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Chapter 4

Oncogenomics/Proteomics of Head and Neck Cancers Howard S. Moskowitz and Jennifer R. Grandis

Abstract  Head and neck cancer treatment has experienced great advances in surgical techniques, radiation therapy, chemotherapy, and molecular targeted strategies through the years. In addition, there has been explosive growth in our understanding of tumor biology through research focusing on individual genes and their gene products. However, poor overall survival persists despite this progress, in large part because treatment decisions continue to be based on traditional parameters, such as tumor size, tumor site, and presence of regional or distant metastases. Head and neck cancer represents an extremely heterogeneous disease with dysregulation of multiple interrelated cellular pathways, including differentiation, apoptosis, angiogenesis, and metastasis. The complexity of interactions between genes and proteins and the environment and the difficulty of finding the right combinations of targets to study pose fundamental problems with successful identification of therapeutic targets and predictive elements. Oncogenomic and proteomic analyses offer the opportunity to accelerate the pace of discovery for clinically relevant targets. A variety of high-throughput technologies including expression profiling and mass spectrometry technologies are being used to analyze cancer genomes and proteomes with the ultimate goal of identifying new cancer genes and therapeutic targets. Potentially, the identification of diseaseassociated proteins and protein signatures could be used as tumor markers for early detection, response to therapy, or relapse. A greater understanding of the molecular events underpinning clinical outcomes will provide useful tools in the identification of new targets for future therapy. These advances have already begun to manifest in several key areas of treatment including early detection of cancer, evaluation of surgical margins, determination of necessary extent of surgery, and predictions of outcome and recurrence. In this chapter we review key technological advances leading to J.R. Grandis (*) Eye & Ear Institute, Suite 500, 200 Lothrop Street, Pittsburgh, PA 15213, USA e-mail: [email protected]

these recent changes as well as many of the studies helping to implement these technologies and apply them to patients with head and neck cancer. Keywords  Oncogenomics • Proteomics • Microarray • Gene expression

Introduction Head and neck squamous cell carcinoma (HNSCC) is the most common histology of cancers arising from the upper aerodigestive tract, comprising approximately 90% of all tumors. HNSCC encompasses a variety of subsites, but despite possessing similar histologic characteristics, the clinical behavior, such as metastatic rate and response to therapy, varies between subsites and even within an individual subsite, indicating biologic heterogeneity in the setting of common histology. Current treatment strategies rely on traditional clinical, radiologic, and histopathologic parameters to determine the stage of disease using the T (tumor), N (node), and M (metastasis) classification system. This system allows for the estimation of disease burden which is presumed to predict clinical outcomes and assist the clinician in making the most appropriate decision for patient management. However, the biologic heterogeneity of HNSCC is reflected by the dysregulation of multiple pathways, including cellular differentiation, angiogenesis, and apoptosis. Apparently, identical histologic tumors may have similar phenotypic characteristics, but develop through dysregulation of different pathways and can have different clinical courses. Despite their intrinsic differences, all HNSCCs are treated similarly. Standard therapy for stage I/II tumors is surgical resection and/or radiation therapy. By contrast, treatment for advanced stage III/IV tumor requires the combination of chemotherapy, radiation therapy, and/or surgery. Given this relatively uniform treatment, clinical outcome after curative therapy varies greatly. The advent of new surgical techniques, radiation therapy, and chemotherapy have improved local

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_4, © Springer Science+Business Media, LLC 2011

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control and overall quality of life, but survival rates for head and neck cancer have not increased significantly. It is likely that the diversity in outcome reflects intrinsic heterogeneity in the molecular components of individual tumors. Clinical outcome has not been shown to be accurately predicted by clinical, radiographic, or histologic characteristics. A limited number of histologic features, such as perineural, perivascular, or nodal extracapsular spread are associated with increased tumor aggressiveness and may influence clinical care. Unfortunately, currently recognized individual markers associated with tumor development generally lack sensitivity or specificity and there is currently no single molecular marker that is used for patient management in HNSCC. Human papilloma virus (HPV) may ultimately serve as a putative marker for HNSCC as it is more common in nonsmokers and nondrinkers and these tumors have a better outcome independent of the specific therapy. Given the variety of molecular changes found in these tumors, a greater understanding of the molecular basis of the biochemical pathways involved in carcinogenesis potentially can facilitate diagnosis, drug discovery, and therapy for affected patients. These molecular changes involve interacting networks that operate at the transcriptional, translational, and posttranslational levels. Traditional approaches have generally not been useful due to the complexity of interactions, the difficulty of finding the proper combinations of genes and proteins to investigate, and the reliance on techniques that examine one or only several genes or proteins at a time. The application of novel unbiased discovery technologies offers the opportunity for comprehensive and systematic molecular analysis to capture the complex cascade of events underpinning the clinical behavior of tumors. Tumors are believed to have molecular footprints that can be identified through the combined application of high-throughput profiling techniques and sophisticated bioinformatics tools for complex data analysis and pattern recognition. The main underlying goal is the identification of new targets that may provide insights into the underlying mechanisms of cancer biology, which in turn can potentially lead to novel approaches to cancer diagnosis, prediction of clinical outcomes, and development of new therapeutic targets.

Oncogenomic Technologies Cancer can be simplistically thought of as the overexpression of oncogenes and/or the silencing of tumor suppressor genes. However, in most cancers, including HNSCC, cancer development and progression is likely due to numerous genetic alterations involving a variety of different pathways. Although common alterations underlie many types of cancer,

H.S. Moskowitz and J.R. Grandis

an individual cancer often develops due to an accumulation of specific mutations in DNA. Since these mutations accumulate randomly, different combinations of mutations exist between different individuals with the same type of cancer. Through the years, the cytogenetic analysis of cells has evolved from the gross visual analysis of chromosomes to a detailed analysis of the regions of chromosomal gain, loss, and translocation. These techniques include comparative genomic hybridization (CGH) where normal and tumor DNA is labeled and hybridized to normal metaphase chromosomes and the fluorescence pattern is then analyzed for increased or decreased intensity representing copy number differences between genomes. Similarly, fluorescent in situ hybridization (FISH) utilizes labeled sequence specific probes, allowing for the detection of particular genes of interest as well as visualization of copy number per cell. More localized and specific analysis has been made possible through the advent of high-throughput DNA sequencing facilities as well as novel approaches to look at genomic variability. Single nucleotide polymorphisms (SNPs) are areas in the genome with an altered DNA sequence that may be markers for disease predisposition or may be used to genetically identify patients. Microsatellites are tandem nucleotide repeats that are generally located in noncoding areas of the genome. They can have variable length and have been mapped to specific chromosomal regions, allowing for the detection of adjacent genes of interest. In addition, microRNAs are a noncoding family of genes involved in posttranscriptional gene regulation that are associated with cell proliferation, cell differentiation, cell death, and carcinogenesis. Each of these can be investigated through the use of array technology. The most commonly utilized platform for oncogenomic analysis currently is DNA microarray technology, which offers the capacity for parallel measurement of relative gene expression levels (Fig. 4.1). These technologies are based on the selective mRNA or cDNA hybridization to DNA probes on the array surface. There are two general categories of microarrays, commercially available microarrays with defined content or microarrays produced with variable and customizable content. Microarray technology involves DNA sequence hybridization onto microscopic surfaces, which can be read by a laser able to detect the signal of minute fluorophores. These studies can incorporate nearly the entire known genome in a single experiment. Each of these technologies generates large amounts of data that can be generated from a single sample, particularly from tumor lysate or serum. Bioinformatics technologies enable the statistical analysis of this data and address the issues of data management and generate prediction algorithms to shortcut the experimental process. These data can be examined via unsupervised analysis using data based only on gene expression pattern regardless of the specific

4  Oncogenomics/Proteomics of Head and Neck Cancers Fig. 4.1  Algorithm for using DNA microarray analysis to identify altered expression levels in HNSCC. After careful selection of patients, tissue samples are collected from study participants and mRNA is isolated. The mRNA represents the expression profile of the isolated cells as only active genes will produce mRNA. Microarray data from various tissues can be compared to generate differential expression patterns reflective of variations in gene expression between subjects. This data can be combined to define cancer signatures reflective of specific steps in tumorigenesis

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1) Samples of tissue are collected from appropriate areas to be studied

No tumor

HNSCC

2) mRNA is isolated Genes from no tumor tissue sample ON

mRNA

OFF

ON

mRNA

Genes from HNSCC tissue sample ON

mRNA

ON

OFF

mRNA

3) DNA copies are generated and labeled with various markers/probes 4) Labeled samples are applied to the microarray 5) Microarray is scanned and data is collected 6) Data processing, normalization, and differential expression analysis 7) Meta-profiles and cancer signatures

characteristics of the tissue being examined. This approach offers the potential to segregate different tumor types and allows identification of tumor subtypes that are not distinguishable by clinical, radiologic, or histologic characteristics. By contrast, supervised approaches select genes with parameters or conditions and the analysis is dependent on the supervising parameter to discriminate the groups or categories with highest prediction accuracy. A predictive gene list is generated from a training set and the results are then confirmed by cross validation and analysis by an independent cohort of patient samples.

Proteomic Technologies Proteome analysis is complementary to DNA microarray technology. Some techniques of proteomic analysis are widely used and clinically applicable, such as enzyme-linked immunosorbent assay and immunohistochemistry, while others are used primarily as research tools, such as immunoblotting and immunoprecipitation. Most of these techniques are limited to the study of only one or a few proteins at a given time. More comprehensive screening is permitted through 2D gel electrophoresis (2-DE). 2-DE is the method with highest resolution for the separation of protein mixtures and is believed to be superior for pattern analysis of complex samples. However, 2-DE may be difficult to use with certain proteins such as membrane proteins and basic proteins and

has limited resolution of proteins in the low molecular weight spectrum. 2-DE separates proteins according to isoelectric points (isoelectric focusing) followed by separation according to molecular mass (SDS-PAGE). Peptide mass fingerprinting permits in-gel digestion of the protein spot of interest with a specific enzyme and resulting peptides are extracted from the gel and molecular weights of these peptides are measured. Alternatively, the peptides can be fragmented in a mass spectrometer yielding partial amino acid sequences from the peptides which act as sequence tags. Fundamentally important to recent advances in proteomics have been improvements in the speed, accuracy, and sensitivity of mass spectrometry (MS) instruments for the analysis of complex protein mixtures or tissues (Fig. 4.2). MS analyzes proteins or peptides as ions which can be distinguished based on mass to charge ratio (m/z). Basic components of the instrument are the ion source which volatilizes and ionizes the proteins, mass analyzer which separates proteins based on m/z values, and the detector which detects the sample after separation. The two most commonly used MS approaches are matrix-assisted laser desorption ionization (MALDI) and surface-enhanced laser desorption ionization (SELDI). These new high-throughput methodologies have the ability to observe large numbers of protein events. Furthermore, as compared to 2-DE they permit improved speed, high-throughput capability, lower amounts of protein sample, effective resolution of low mass proteins, and direct application to assay development. Furthermore, sample loading and processing can be fully automated.

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Ionization

Mass Analyzer

Non-tumor

Detector

Tumor *

* *

Protein Fingerprint

Fig.  4.2  Mass spectrometry approaches to biomarker analysis. Analysis begins with a protein or peptide mixture that is processed to maximize the number of detected differentially expressed proteins. The sample is subsequently ionized by a variety of instruments such as a laser and separated by a mass analyzer (TOF or ion trap) based on mass and charge. The resulting spectra are representative of the ionized proteins within the initial sample. Bioinformatics approaches are then utilized to compare the spectra to identify unique and differing protein components (asterisk indicates differentially expressed m/z species)

MALDI is commonly used for bioanalysis and employs laser energy to ionize and volatize proteins. A matrix such as a UV absorbing organic acid is mixed with the sample to absorb laser energy and transfer it to the proteins to generate ions which are then transferred to the mass analyzer. Ionization is not uniform and depends on relative protein abundance and intrinsic chemical characteristics. MALDI is generally coupled with a time-of-flight (TOF) mass analyzer which separates proteins based on time to traverse a flight tube and strike a detector. MALDI-TOF-MS is a particle counting method that relies on molar abundance. It requires minimal sample preparation, can distinguish hundreds to thousands of proteins from a complex mixture, and can detect subtle protein modifications. However, MALDI has a limited mass range, limited sensitivity for low abundance proteins, and proteins with extremely high concentration can interfere with detection of proteins with similar m/z ratios. SELDI utilizes a surface to capture and partially purify proteins from a complex sample based on physical and biochemical properties and is dependent on protein conformational stability for reliable detection. A variety of coated surfaces are presently available that bind proteins based on hydrophobicity, anionic or cationic charge, or binding to metals. SELDI also partially purifies the protein sample,

making it less complex than the similar unfractionated sample for MALDI. This partial purification may lose critical proteins, but theoretically generates fewer problems with highly abundant proteins. When the process is expanded to many hundreds of samples, population-specific protein expression profiles can be deduced that are characteristic of the assayed group. However, the identified mass spectrum does not enable protein identification and none of the interactions are specific. Reverse phase protein arrays (RPPA) are another highthroughput platform for marker screening. RPPA utilizes lysed histopathologically relevant pure cell populations. The lysate is immobilized in an array configuration via a pinbased microarrayer onto nitrocellulose slides with each spot containing the whole cellular protein contents. Each slide is then probed with an antibody that can be detected by a variety of assays. Protein samples are arrayed in miniature dilution curves to ensure that the analyte of interest remains in the linear range of detection. Finally, tissue microarray (TMA) technology applies advanced array-based approaches to data gathering with standardized medical pathology laboratory practices. A TMA block is loaded with freshly sectioned core biopsies from paraffin-embedded tissues from large cohorts of cancer patients on a single slide. Automated digital image capture is followed by pathologist scoring of the image. Further evolution in the analysis of stained TMA sections involves automated scoring of staining intensities and features on TMA slides using image analysis software. TMA provides the capability to perform rapid analysis of comprehensive panels of normal and disease specimens. TMA allows visualization of molecular targets in thousands of tissue specimens at a time and reveals cellular localization, prevalence, and clinical significance of candidate genes and gene products. However, TMA is limited by the availability of antisera, only provides a qualitative estimation of protein levels, and may miss important histologic areas due to the small size of the core biopsies utilized in these arrays.

Oncogenomics of HNSCC Genomic Changes Underlying Malignant Transformation Cancer develops from the accumulation of various genetic alterations. DNA microarrays have had a major impact on biomedical research and have emerged as a powerful tool for the parallel measurement of relative gene expression levels and have been widely studied in HNSCC (Table 4.1). The usage of DNA microarrays to generate clinically relevant molecular

4  Oncogenomics/Proteomics of Head and Neck Cancers Table 4.1  HNSCC studies incorporating DNA microanalysis Authors Tissue samples Array platform Jeon et al. [1]

Jarvinen et al. [3]

Jarvinen et al. [4]

Mendez et al. [5]

Ha et al. [6] Chen et al. [7] Ginos et al. [8]

Ziober et al. [9] Kondoh et al. [10]

Schmalbach et al. [12] Belbin et al. [13] Roepman et al. [14]

Hensen et al. [15]

Rickman et al. [16] Chung et al. [17]

Cromer et al. [18]

25 HNSCC cell lines 1 immortalized oral keratinocyte line 10 HNSCC cell lines 10 primary laryngeal tumors 5 LN+, 5 LN− 18 oral tongue SCC cell lines 19 primary, 7 recurrent OC 8 LN+, 18 LN− 2 premalignant lesions 7 primary HNSCC 7 H&N dysplastic lesions 171 oral SCC 17 dysplastic lesions 25 primary, 16 locally recurrent 19 LN+, 21 LN−, 1 LN unknown 13 oral SCC 3 LN+, 10 LN− 27 oral SCC 5 LN+, 21 LN−, 1 LN unknown 19 leukoplakias 20 primary OC/OP 13 LN+, 7 LN− 4 oral SCC cell lines 9 primary OC All LN + Predictor set: 82 primary OC/OP Validation set: 22 primary OC/OP 55 LN+, 49 LN− 22 primary HNSCC 11 LN+, 11 LN−

186 primary HNSCC 132 LN+, 50 LN−, 4 LN unknown 55 primary HNSCC, 5 recurrent 26 LN+, 14 LN−, 20 LN unknown 34 primary HP SCC 30 LN+, 4 LN−

Incyte Genomics Human GEM2 cDNA Agilent Human 1A

Agilent Human 1A and Human 1A (v2) Affymetrix Test-1 and HuGeneFL

Affymetrix Hu95A. v2 Affymetrix U133 2.0 Plus Affymetrix HG-U133A

Affymetrix HG-U133A IntelliGene HS Human

Affymetrix HG-U95Av2 Custom cDNA microarray Qiagen Human Array-Ready Oligo set (version 2.0) Qiagen Human Array-Ready Oligo set (version 2.0) Affymetrix HGU133_Plus2 Agilent Human 1

Affymetrix HG-U95A

signatures has grown in its acceptance. Early studies showed the heterogeneous nature of HNSCC tumors at the molecular level. However, direct comparison between studies has often proved difficult due to the variety of gene-expression arrays, platforms, and data analysis algorithms used.

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Preclinical work in HNSCC cell lines provided initial insights into the genetic variations that may underlie the cancer phenotype. Cell lines offer relative homogeneity of samples for investigation, but may suffer from artifacts of immortalization and passage in vitro compared with human tumors. One microarray study analyzed 25 HNSCC cell lines and one immortalized human oral keratinocyte cell line and found wide alteration in the gene expression in cell cycle regulation, oncogenesis, cell proliferation, differentiation, and apoptosis [1]. Their work revealed two distinctive subtypes of gene expression patterns, but these patterns did not seem to correlate with the clinical staging or differentiation grade of the original tumors. A more recent study used SNP-array based loss of heterozygosity (LOH) profiling on whole-genome loss of 41 HNSCC cell lines and found several frequent LOH regions [2]. This study identified a region on chromosome 8 that exhibited the most frequent LOH (87.9%) and found that the mitochondrial tumor suppressor gene 1, a candidate tumor suppressor gene residing in this area, was consistently down regulated in expression, suggesting it may be a tumor suppressor in HNSCC. Another report utilized genome-wide CGH and expression microarray analyses to reveal known and novel amplicons that showed concomitant increase of copy number and expression of target genes for both laryngeal SCC cell lines and primary tumors [3]. They found that the overexpression of 739 genes could be attributed to gene copy number alteration in cell lines, of which 325 genes showed the same phenomenon in primary tumors. Subsequently, this group analyzed oral tongue SCC cell lines and found that these cell lines exhibited similar genomic alterations as had been previously been found in their laryngeal SCC cell lines despite the differences in clinicopathologic features between these anatomic subsites [4]. A wide variety of genes were found to be altered including deletions of known tumor suppressor genes such as FHIT, CSMD1, and CDKN2A. Other studies have attempted to provide a framework for improving our understanding of the molecular events underpinning various aspects of these tumors. The progression of normal epithelia through premalignancy to HNSCC is a multistep process that has been associated with distinct histologic characteristics at each stage. An early study analyzed invasive SCC lesions from the oropharynx and oral cavity and using hierarchical clustering analysis they were able to show that oral SCC was distinguishable from normal oral tissue, but there was heterogeneity among the tumors even of a particular histopathologic grade and stage [5]. This study identified 239 genes that were overexpressed and 75 genes that were down regulated, but could not find statistically significant differences in gene expression between metastatic and nonmetastatic tumors. Later, another group established

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a transcriptional progression model of HNSCC in the progression from normal mucosa to dysplastic epithelium to invasive HNSCC [6]. Matched samples were analyzed using gene expression arrays, significance analysis of microarrays, hierarchical clustering, and principal components analysis to identify genes with differential expression patterns between the tissue groups. The progression from normal to premalignant was associated with altered expression of 334 genes (108 up regulated and 226 down regulated) while the progression of premalignant to malignant was only associated with altered expression of 18 genes (5 up regulated and 13 down regulated). This transcriptional model suggested that the majority of alterations occurred before the development of invasive cancer. An alternative strategy was used in another study employing forward and stepwise logistic regression analyses to identify potential biomarkers for the early detection of oral SCC by comparing gene expression of primary oral SCC, oral dysplasia, and clinically normal oral tissue [7]. They identified combinations of genes which differentiated oral SCC from controls that included laminin-gamma2 chain, collagen type IV alpha1 chain, collagen type I alpha1 chain, and peptidyl arginine deiminase type 1. Another group analyzed 41 HNSCC tumors from various anatomic sites and compared them with normal oral mucosa with gene expression arrays [8]. They used statistical and data filtering criteria to identify 2,890 genes differentially expressed between the two groups and revealed functional gene expression signatures that were highly represented in HNSCC, including those involved in inflammatory response, epidermal differentiation, cell adhesion, and extracellular matrix functions. They suggested that the disease signature is an intrinsic feature of a HNSCC and may function as a predictor of early local treatment failure. Several studies have attempted to build on the growing lists of putative biomarkers by generating gene sets which may be able to lead to useful predictions regarding the propensity for a given lesion to be or develop into a cancerous lesion. One study matched tumor and normal specimens from the oral cavity and analyzed microarray gene expression data with a supervised learning algorithm [9]. This study generated a 25-gene signature that could classify normal and tumor specimen that was highly accurate on independent validation test sets, but failed to predict nonoral tumors. Many of the genes in the predictor set had been previously implicated in oral SCC. The predictor set comprised several epithelial marker genes that had categories of potential interest including extracellular matrix components and cell adhesion molecules. Similarly, a different group attempted to generate a classifier set for oral SCC and leukoplakias and found differential expression of 118 marker gene candidates by complementary DNA microarray [10]. Further evaluation demonstrated an 11-gene predictor set that could distinguish the two groups with greater than 97% accuracy.

H.S. Moskowitz and J.R. Grandis

Genomic Changes Underlying Metastases Metastasis is the principal cause of death in patients suffering from cancer, but the underlying molecular mechanisms are poorly understood. It is widely believed that the accumulation of genetic damage leads to the expression of a malignant phenotype that precedes metastasis formation. The reliable detection of the presence of metastases in local lymph nodes to distinguish metastatic from nonmetastatic tumors would be important for treatment planning. One study compared the gene-expression profiles of adenocarcinoma metastases of multiple tumor types to unmatched primary adenocarcinomas [11]. They found a gene-expression signature of 17  unique genes that could distinguish the two groups. Furthermore, an analysis of 279 independent primary solid tumors of diverse types and organ sites revealed that tumors carrying this signature were associated with metastasis and poor clinical outcome. With specific regard to HNSCC, several groups have investigated differences in gene expression between primary tumors that had or had not metastasized. In one analysis of tumors from the oral cavity and oropharynx, 101 genes demonstrated significant expression differences between the metastatic and nonmetastatic tumors [12]. These genes included a variety of cellular functions putatively associated with cancer behavior and the gene with the greatest differential expression between the metastatic and nonmetastatic tumors was collagen type 11 alpha1. A different study used microarray analysis to measure gene-expression changes associated with tumor progression in patients with stage III or stage IV untreated oral SCC [13]. They identified 140 genes that consistently increased in expression during the progression from normal tissue to invasive tumor to metastatic node as well as 94 genes that decreased in expression in a similar progression, which revealed a distinct pattern of gene expression during the progression from histologically normal tissue to primary carcinoma to nodal metastasis. In another study, 82 primary tumors located in the oropharynx or oral cavity regions were analyzed using DNA microarray gene-expression profiling [14]. This study established a set of 102 predictor genes for determining the presence of lymph node metastases. Many of the predictor genes they found were previously implicated in metastasis. The application of this gene set to a validation group gave an overall predictive accuracy of 86% as compared with 68% based solely on clinical diagnosis. A subsequent study implemented this dataset as a reference dataset and an independent gene expression dataset of metastasized and nonmetastasized HNSCC tumors as a validation dataset [15]. They utilized supervised gene-based and pathway-based analysis to evaluate differences in gene expression to enhance the understanding of the biological context of the results. The identified gene sets were involved in extracellular matrix remodeling

4  Oncogenomics/Proteomics of Head and Neck Cancers

(including matrix metalloproteinases (MMPs) and their regulatory pathways) as well as hypoxia and angiogenesis. Another group looked at 186 primary tumors and analyzed the samples with respect to whether the development of metastasis was the first recurrent event [16]. They collected transcriptome and array-CGH data followed by nonsupervised hierarchical clustering to distinguish tumors differing in pathological differentiation. They were able to identify associated functional changes and created a fourgene model (PSMD10, HSD17B12, FLOT2, and KRT17), which predicted the metastatic status with 77% success in a separate validation group and the prediction was independent of clinical criteria. Similarly, another study revealed that gene expression patterns in 60 primary and previously untreated HNSCC allowed the tumors to be categorized into four distinct subtypes with statistically different recurrencefree survival [17]. Clinical nodal staging resulted in low prediction accuracy when used as the supervising parameter. However, supervised analyses using pathological staging to predict lymph node metastasis status improved the prediction accuracy of gene expression from the primary tumor which was further improved by analysis based on anatomic subsites leading to a prediction accuracy of 83%. A large-scale gene expression analysis of the hypopharynx, a location associated with particularly aggressive behavior, found 119 genes that were highly differentially expressed between early and late tumors [18]. Furthermore, 164 differentially expressed genes were found that differentiated between relatively nonaggressive and aggressive tumors. Clustering of the associated probe sets defined the two groups of samples and correctly assigned 92% of the tumors. In a separate study, genome-wide analysis was performed looking for LOH and allelic imbalance (AI) on specimens of tumor stroma and tumor epithelium isolated by laser capture microdissection on 122 patients with HNSCC and a history of smoking [19]. They found nearly twice as many areas of LOH/AI within the stroma as was found in the epithelium, more than 40 areas in total. Furthermore, they found three stroma-specific loci that were significantly associated with tumor size and cervical lymph node metastasis, highlighting the importance of examining stromal and epithelial elements and suggesting that stromal alterations play an important role in HNSCC behavior.

Genomic Changes Underlying Variable Responses to Treatment Treatment protocols often involve the use of chemotherapy and/or radiation therapy. Several recent studies have directed their attention toward the identification of genetic alterations that would give prognostic information regarding a given

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tumor’s likelihood of response to various treatment protocols. One study on HNSCC cell lines that exhibited relative radioresistance and radiosensitivity identified 167 genes that were significantly overexpressed in radioresistant cells, 25 of which included cancer-related genes involved in growth, proliferation, apoptosis, and adhesion [20]. Another study used significance analysis of microarrays for gene selection and a multivariate linear regression model for the prediction of radiosensitivity [21]. They identified three novel genes whose expression values correlated with radiation sensitivity and the overexpression of one of these genes, RbAp48, in cancer cells lines induced radiosensitization. In another recent study, 92 biopsies were obtained from untreated HNSCC patients prior to treatment with cisplatinbased chemoradiation for advanced HNSCC [22]. This group utilized supervised analyses to predict locoregional control and disease recurrence and found several gene sets that were enriched in recurrences. Furthermore, they utilized a signature established by Chung et al. [17] for HNSCC defining a high-risk group and found it to be predictive for locoregional control and disease-free survival in their dataset. Finally, a more targeted analysis utilized a cDNA array consisting of genes associated with angiogenesis and/or metastasis [23]. Seventeen genes were correlated with locoregional failure, of which MDM2 and erbB2 were found to be predictors of locoregional failure in their population of patients treated with chemoradiation therapy.

Genomic Changes Found in Surrogate Tissues An evolving area of investigation involves the use of surrogate tissues in the investigation of HNSCC. Using saliva from patients with primary T1/T2 oral SCC with matched control patients in terms of age, gender, and smoking history, one group used microarrays to profile the human salivary transcriptome [24]. They found 1,679 genes that were significantly differentially expressed between the groups including seven cancer-related mRNA biomarkers that exhibited at least a 3.5-fold elevation in oral SCC saliva (IL8, IL1B, DUSP1, HA3, OAZ1, S100P, and SAT). The combination of four of these biomarkers had a discriminatory power of 91% sensitivity and specificity for oral cancer detection. A subsequent study compared the clinical accuracy of saliva with that of blood by using RNA biomarkers for oral cancer detection [25]. Using four serum mRNA markers, a sensitivity of 91% and a specificity of 71% were obtained for distinguishing oral cancer. However, the four salivary mRNA markers had a higher receiver operating characteristic curve value demonstrating that for oral cancer detection, salivary transcriptome diagnostics may demonstrate a slight advantage as compared with serum.

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Meta-Analyses of HNSCC Microarray Studies Given the increasing number of studies of the HNSCC transcriptome, a recent analysis looked at the studies incorporating DNA microarray analysis to examine genetic expression changes associated with the development of HNSCC [26]. Eighty-four genes were identified with common alterations in transcriptional expression across multiple studies. Many of these had been reported to be involved with HNSCC, including MMPs, integrins, collagens, fibronectin, tenascin C, and cathepsin L, as well as many genes with less characterized roles in HNSCC. Only one gene, transglutaminase 3 was common to at least three of the reviewed studies. Overall, they found that genes encoding extracellular matrix and integral membrane proteins, cell adhesion molecules, and proteins involved in epidermal development and differentiation were most frequently identified in these studies. Furthermore, their results suggested a global down regulation of genes encoding ribosomal proteins and cholesterol biosynthesis enzymes and an up regulation of MMPs and inflammatory response genes. Another study looked at 63 HNSCC transcriptomic studies in three categories of comparisons, premalignant vs. normal (Pre), primary tumors vs. normal (TvN), and metastatic or invasive vs. primary tumors (Meta) [27]. They used a systemsbiology approach via network-based meta-analysis and verified that 82 genes, 1,260 genes, and 321 genes in the Pre, TvN, and Meta comparisons, respectively, were found reported at least twice. Overall, 1,442 unique genes were reported at least twice in the studies that they analyzed. In terms of the direction of fold changes of the verified genes, the least contradiction was found in the TvN group and the most contradiction was found in the Pre group. Furthermore, they found that few genes overlapped between the Pre and Meta groups although many genes overlapped between the other pairs of comparisons. Genes that were highly reported in prior studies across all three stages were ECM1, EMP1, CXCL10, and POSTN. Subsequently, they constructed knowledge-based networks which revealed that integrin signaling and antigen presentation pathways were highly enriched in the dataset and they found that chromosomal regions of 6p21, 19p13, and 19q13 had genomic alterations that were correlated with the nodal status of HNSCC.

Proteomics of HNSCC Tumor Tissue Studies High-throughput proteomic technologies have been utilized to detect biologically significant differences in protein expres­ sion of HNSCC in the same types of samples utilized in

H.S. Moskowitz and J.R. Grandis

gene-expression analysis. These studies have used a variety of techniques as outlined earlier in the chapter. One study utilized SELDI-TOF-MS to generate proteomic spectra and used the “Lasso algorithm” to extrapolate proteomic patterns that can best discriminate HNSCC patients from noncancer controls which identified 65 significant data points to be used for discrimination [28]. Testing of these points yielded moderate sensitivity of 68% and specificity of 73% indicating that with further improvement and validation it may be useful as a screening test for HNSCC in the future. More recently, another study analyzed 113 HNSCC, 73 healthy, 99 tumor-distant, and 18 samples of tumor-adjacent squamous mucosa by SELDI-TOF-MS [29]. They found 48 protein peaks differentially expressed between healthy mucosa and HNSCC. A supervised prediction analysis revealed greater than 90% classification of healthy mucosa and tumor samples and 72% of the tumor-adjacent mucosa samples were predicted as aberrant, providing evidence for the existence of genetically altered fields with inconspicuous histology. MALDI-TOF has also been successfully used in HNSCC proteomic studies. In one such investigation, MALDI-TOF was coupled with magnetic bead fractionation to analyze an HNSCC cohort consisting of matched pretreatment and 6–12 month posttreatment samples for analysis [30]. A set of approximately 200 spectral peaks was used and was able to largely correctly classify normal from pretreatment HNSCC samples, pretreatment from posttreatment, and normal from posttreatment samples. This showed the potential for use of this technology as a discovery platform in order to generate biomarker panels that potentially could be used for more accurate prediction of prognosis and treatment efficacies for HNSCC. Another study used multidimensional LC–MS/MS to identify proteins that are differentially expressed in HNSCC for cancer biomarker discovery [31]. More than 811 proteins were identified, which included structural proteins, signaling components, and transcription factors. They utilized a panel of the three best performing biomarkers, YWHAZ, stratifin, and S100-A7, to discriminate cancerous from noncancerous head and neck tissue. Their differential expression was verified by immunohistochemistry, immunoblotting, and RT-PCR and achieved a sensitivity of 92% and specificity of 87% in an independent set of HNSCC in discriminating tissue types. More recently, an analysis of samples from HNSCC patients with 2-DE and MALDITOF-MS revealed 181 proteins with differential expression between pretreatment and posttreatment samples [32]. Classification by disease status revealed significant differential expression of 16 proteins including several protease inhibitors and other molecules with direct implications on tumor survival. Another study attempted to validate DNA microarray results on a subset of genes that could potentially

4  Oncogenomics/Proteomics of Head and Neck Cancers

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serve as biomarkers of oral SCC [33]. This group was able to validate five of six potential biomarkers by using qRT-PCR to examine expression changes in oral SCC and normal control tissues. TMA analysis then revealed that four of the six biomarkers (SPARC, POSTN, TNC, and TGM3) had differential expression and localization.

with newly diagnosed T1 or T2 oral cavity or histologically confirmed oropharyngeal SCC. Their analysis revealed that IL-8 was detected at higher concentrations in saliva and IL-6 was detected at higher concentrations in serum of patients with oral SCC, indicating that these markers and tissues hold promise for biomarker analysis in oral SCC [38].

Surrogate Tissue Studies

Challenges of Oncogenomics/ Proteomics in HNSCC

Serum studies have been widely used in the investigations of HNSCC given the challenges in obtaining repeat tumor samples. One study used MALDI on sera from 99 HNSCC and 143 controls to obtain serum protein patterns [34]. The mass spectra and linear discriminant analysis were used to select the top 45 spectral features. The subsequent spectral profiles from the sera of the HNSCC patients statistically significantly differed from the sera of control subjects. In a separate study, samples were analyzed by SELDI-TOF and 80 common peaks or clusters were generated from the training set and used to create classification trees [35]. This algorithm correctly identified 91% of HNSCC sera in the training set and 83% of HNSCC samples in the test set, yielding an overall sensitivity of 83% and an overall specificity of 90%. Furthermore, they were able to identify a particular peak as the known biomarker metallopanstimulin-1 based on mass and whose relative intensity consistently correlated with levels detected by radioimmunoassay. More recent research has sought novel surrogate tissue sources, which may be convenient for investigation. Alterations in the levels of biomarkers have been investigated in other body fluids that are near or bathe tumor sites. Accordingly, saliva is an ideal complementary resource for developing HNSCC diagnostics and more recent study attempts have focused on the use of salivary proteomics for oral cancer biomarker discovery. One analysis collected saliva from 64 oral SCC and 64 healthy subjects and utilized subtractive proteomics to find that several salivary proteins were differentially expressed [36]. Five candidate biomarkers were validated and demonstrated high sensitivity (90%) and specificity (83%) in detecting oral SCC. Another recent study, found two proteins, alpha-1-B-glycoprotein and complement factor B proteins, to be present in patients with HNSCC but not in normal specimens, while cystatin S, parotid secretory factor, and poly-4-hydrolase beta-subunit proteins were detected in most normal saliva but not in HNSCC [37]. These results suggest that certain proteins are differentially found in saliva from patients with HNSCC and a small set of proteins may be useful for future validation for clinical investigation. Finally, another study built on prior data indicating that the expression of IL-6 and IL-8 are uniquely associated with oral SCC. They analyzed patients

The application of these novel technologies offers many opportunities for advanced analyses of HNSCC. With the completion of the Human Genome Project and advances in array technology, gene expression studies offer an opportunity to look at the full complement of genes expressed by a tumor. Gene expression profiling experiments have generated a tremendous amount of information regarding concomitant genetic events during disease. However, the functional consequences of disease are also regulated by the deregulation of protein products and protein networks so that the information flow cannot be ascertained from gene analysis alone.

Key Advantages and Limitations of DNA Microarrays: Advantages: −− Provide insight into fluctuations in gene transcription. −− Capable of generating large amounts of expression data quickly. −− Current microarrays give expression data from essentially the entire genome. −− Technological advances have generated microarrays that can be implemented using automated, high-throughput strategies, at reduced costs. Limitations: −− High quality RNA is required for the generation of good expression data. −− Changes in RNA expression may not correlate with changes in protein levels. −− Advanced biostatistics are necessary to process vast amounts of data generated.

 Furthermore, there are a variety of potential pitfalls in microarray analysis that may obscure the quantification of genes of interest. One of the most important variables relates

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to the quality of the transcripts utilized for the microarray which may relate to initial and long-term tissue handling as well as processing of the transcripts for use in the microarray studies. A recent report indicated that there may be a storagetime decrease in the predictive performance of tissue samples [1]. Other common causes of signal variations include errors with fluidics protocols, spoiled or omitted hybridization cocktail reagents, and inaccurate quantification of labeled samples. There are also a variety of factors inherent to the microarray technology such as intensity-dependent dye effect and spatial-dependent dye effect that can influence the quantification process. In addition, studies vary in the heterogeneity of the cell types included in the samples from 50% tumor cells to the pure isolation of single tumor cells. By contrast, the study of disease-related protein changes provides unique challenges since these changes are dependent on highly regulated processes at the transcriptional, translational, and posttranslational levels.

Key Advantages and Limitations of Proteomic Approaches: Advantages: −− Provide insight into fluctuations in transcribed and translated gene products as well as posttranslational modifications. −− Capable of using a variety of tissue sources with minimal processing to analyze variations. −− Increasingly offering high-throughput technologies. Limitations: −− High abundance proteins may obscure data. −− Generally, only analyze a minority of proteins within the entire sample. −− Difficult to correlate individual spectral peaks/signatures with actual proteins.

 Many of the standard proteomic approaches rely on the usage of complex protein mixtures and the indirect assignment of spectra to identify target proteins. These approaches are often hampered by the presence of large quantity proteins that may obscure quantification of the proteins of interest. Accordingly, there has been increasing interest in developing protein microarrays capable of identifying hundreds of protein events simultaneously; however, these arrays have a set of unique problems. Protein interactions are governed by complex associations between the target protein and the ­antigen-binding site on the antibody. Furthermore, proteins tend to denature with changes in pH or temperature and antibodies must exhibit strong affinities and specificity to each of their respective

substrates especially in the analysis of specific protein states, such as phosphorylation or proteolytic cleavage. In addition, the variation in protein concentration in cells may vary widely, so detection methods must exist that can quantify protein concentration over many orders of magnitude. These studies also require careful experiment planning starting with the selection of appropriate controls. Many studies use matched “normal” epithelium, but this may confound interpretations of gene expression changes occurring in HNSCC tumorigenesis. Although logistically difficult to achieve, the theoretically ideal control tissue would match for patient age, gender, smoking and drinking history, and other variables to minimize further confounding factors.

Conclusion and Future Directions The goals of oncogenomics and proteomics are to improve diagnosis, therapy, and cure rates for cancer patients. A patient’s genomic signature of a cancer may serve as the basis for choosing the most effective therapy for the individual patient to improve their chances of recovery and their quality of life. Oncogenomics and proteomics have progressed from molecular profiling to model systems, cancer pharmacology, and clinical trials. Although it is unlikely that a single biomarker accurately detects the presence of HNSCC, analyses that can detect multiple markers may have improved predictive value when used in combination. Imperfect biomarkers may still be clinically useful for serial testing of single individuals because acute changes in biomarker levels may signal the need for an aggressive search for the cause. An important challenge for biomarker validation is the considerable molecular heterogeneity of individual cancers and the low overall incidence of the disease in general population, making it difficult to validate the true prognostic potential of a biomarker or panel of biomarkers. Nonconcordance of predictive gene lists is common in many microarray studies using different platforms and data mining tools and may represent differences in experimental design or data analyses, but also may represent true differences in biology based on different subsites or other unknown factors. Furthermore, although current oncogenomic and proteomic approaches may yield valuable information in the identification of novel diagnostic markers, gene- and proteinexpression profiles may not be able to provide an alternative method of diagnosis on their own. It may become necessary to include other technologies, such as metabolomics, peptidomics, glycomics, and lipidomics for better isolation and identification of molecular targets. In order to obtain reliable prognostic markers, these technologies will need to be combined with advanced bioinformatics tools to integrate and mine the data from basic and clinical research. Once molecular signatures are successfully validated, it will also

4  Oncogenomics/Proteomics of Head and Neck Cancers

be important to perform long-term clinical studies to determine the validity of using these signatures in independent cohorts of patients for the prediction of patient response to therapeutic options.

References 1. Jeon GA, Lee JS, Patel V, et al. Global gene expression profiles of human head and neck squamous carcinoma cell lines. Int J Cancer. 2004;112:249–58. 2. Ye H, Pungpravat N, Huang BL, et al. Genomic assessments of the frequent loss of heterozygosity region on 8p21.3-p22 in head and neck squamous cell carcinoma. Cancer Genet Cytogenet. 2007;176:100–6. 3. Jarvinen AK, Autio R, Haapa-Paananen S, et  al. Identification of target genes in laryngeal squamous cell carcinoma by high-resolution copy number and gene expression microarray analyses. Oncogene. 2006;25:6997–7008. 4. Jarvinen AK, Autio R, Kilpinen S, et al. High-resolution copy number and gene expression microarray analyses of head and neck squamous cell carcinoma cell lines of tongue and larynx. Genes Chromosom Cancer. 2008;47:500–9. 5. Mendez E, Cheng C, Farwell DG, et al. Transcriptional expression profiles of oral squamous cell carcinomas. Cancer. 2002;95: 1482–94. 6. Ha PK, Benoit NE, Yochem R, et al. A transcriptional progression model for head and neck cancer. Clin Cancer Res. 2003;9:3058–64. 7. Chen C, Mendez E, Houck J, et al. Gene expression profiling identifies genes predictive of oral squamous cell carcinoma. Cancer Epidemiol Biomarkers Prev. 2008;17:2152–62. 8. Ginos MA, Page GP, Michalowicz BS, et al. Identification of a gene expression signature associated with recurrent disease in squamous cell carcinoma of the head and neck. Cancer Res. 2004;64:55–63. 9. Ziober AF, Patel KR, Alawi F, et al. Identification of a gene signature for rapid screening of oral squamous cell carcinoma. Clin Cancer Res. 2006;12:5960–71. 10. Kondoh N, Ohkura S, Arai M, et al. Gene expression signatures that can discriminate oral leukoplakia subtypes and squamous cell carcinoma. Oral Oncol. 2007;43:455–62. 11. Ramaswamy S, Ross KN, Lander ES, Golub TR. A molecular signature of metastasis in primary solid tumors. Nat Genet. 2003;33: 49–54. 12. Schmalbach CE, Chepeha DB, Giordano TJ, et al. Molecular profiling and the identification of genes associated with metastatic oral cavity/pharynx squamous cell carcinoma. Arch Otolaryngol Head Neck Surg. 2004;130:295–302. 13. Belbin TJ, Singh B, Smith RV, et al. Molecular profiling of tumor progression in head and neck cancer. Arch Otolaryngol Head Neck Surg. 2005;131:10–8. 14. Roepman P, Wessels LF, Kettelarij N, et al. An expression profile for diagnosis of lymph node metastases from primary head and neck squamous cell carcinomas. Nat Genet. 2005;37:182–6. 15. Hensen EF, De Herdt MJ, Goeman JJ, et  al. Gene-expression of metastasized versus non-metastasized primary head and neck squamous cell carcinomas: a pathway-based analysis. BMC Cancer. 2008;8:168. 16. Rickman DS, Millon R, De Reynies A, et al. Prediction of future metastasis and molecular characterization of head and neck squamous-cell carcinoma based on transcriptome and genome analysis by microarrays. Oncogene. 2008;27:6607–22. 17. Chung CH, Parker JS, Karaca G, et al. Molecular classification of head and neck squamous cell carcinomas using patterns of gene expression. Cancer Cell. 2004;5:489–500.

91 18. Cromer A, Carles A, Millon R, et al. Identification of genes associated with tumorigenesis and metastatic potential of hypopharyngeal cancer by microarray analysis. Oncogene. 2004;23:2484–98. 19. Weber F, Xu Y, Zhang L, et al. Microenvironmental genomic alterations and clinicopathological behavior in head and neck squamous cell carcinoma. JAMA. 2007;297:187–95. 20. Ishigami T, Uzawa K, Higo M, et al. Genes and molecular pathways related to radioresistance of oral squamous cell carcinoma cells. Int J Cancer. 2007;120:2262–70. 21. Torres-Roca JF, Eschrich S, Zhao H, et al. Prediction of radiation sensitivity using a gene expression classifier. Cancer Res. 2005;65: 7169–76. 22. Pramana J, Van den Brekel MW, van Velthuysen ML, et al. Gene expression profiling to predict outcome after chemoradiation in head and neck cancer. Int J Radiat Oncol Biol Phys. 2007;69: 1544–52. 23. Ganly I, Talbot S, Carlson D, et al. Identification of angiogenesis/ metastases genes predicting chemoradiotherapy response in patients with laryngopharyngeal carcinoma. J Clin Oncol. 2007;25: 1369–76. 24. Li Y, St John MA, Zhou X, et al. Salivary transcriptome diagnostics for oral cancer detection. Clin Cancer Res. 2004;10:8442–50. 25. Li Y, Elashoff D, Oh M, et  al. Serum circulating human mRNA profiling and its utility for oral cancer detection. J Clin Oncol. 2006;24:1754–60. 26. Choi P, Chen C. Genetic expression profiles and biologic pathway alterations in head and neck squamous cell carcinoma. Cancer. 2005;104:1113–28. 27. Yu YH, Kuo HK, Chang KW. The evolving transcriptome of head and neck squamous cell carcinoma: a systematic review. PLoS One. 2008;3:e3215. 28. Soltys SG, Le QT, Shi G, et al. The use of plasma surface-enhanced laser desorption/ionization time-of-flight mass spectrometry proteomic patterns for detection of head and neck squamous cell cancers. Clin Cancer Res. 2004;10:4806–12. 29. Roesch-Ely M, Nees M, Karsai S, et al. Proteomic analysis reveals successive aberrations in protein expression from healthy mucosa to invasive head and neck cancer. Oncogene. 2007;26:54–64. 30. Freed GL, Cazares LH, Fichandler CE, et al. Differential capture of serum proteins for expression profiling and biomarker discovery in pre- and posttreatment head and neck cancer samples. Laryngoscope. 2008;118:61–8. 31. Ralhan R, Desouza LV, Matta A, et al. Discovery and verification of head-and-neck cancer biomarkers by differential protein expression analysis using iTRAQ labeling, multidimensional liquid chromatography, and tandem mass spectrometry. Mol Cell Proteomics. 2008;7:1162–73. 32. Gourin CG, Zhi W, Adam BL. Proteomic identification of serum biomarkers for head and neck cancer surveillance. Laryngoscope. 2009;119:1291–302. 33. Choi P, Jordan CD, Mendez E, et  al. Examination of oral cancer biomarkers by tissue microarray analysis. Arch Otolaryngol Head Neck Surg. 2008;134:539–46. 34. Sidransky D, Irizarry R, Califano JA, et al. Serum protein MALDI profiling to distinguish upper aerodigestive tract cancer patients from control subjects. J Natl Cancer Inst. 2003;95:1711–7. 35. Wadsworth JT, Somers KD, Stack Jr BC, et  al. Identification of patients with head and neck cancer using serum protein profiles. Arch Otolaryngol Head Neck Surg. 2004;130:98–104. 36. Hu S, Arellano M, Boontheung P, et al. Salivary proteomics for oral cancer biomarker discovery. Clin Cancer Res. 2008;14:6246–52. 37. Ohshiro K, Rosenthal DI, Koomen JM, et  al. Pre-analytic saliva processing affect proteomic results and biomarker screening of head and neck squamous carcinoma. Int J Oncol. 2007;30:743–9. 38. St John MA, Li Y, Zhou X, et al. Interleukin 6 and interleukin 8 as potential biomarkers for oral cavity and oropharyngeal squamous cell carcinoma. Arch Otolaryngol Head Neck Surg. 2004;130:929–35.

Chapter 5

Genetics and Epigenetics of Head and Neck Cancer Richard J. Shaw and Jagtar Dhanda

Abstract  Cancer is caused by a multi-step progression of genetic and epigenetic aberrations resulting in a clonal expansion of cells. These cells have a selective growth advantage characterised as the “hallmarks of cancer” including loss of control of the cell cycle, genomic instability, inhibition of apoptosis, insensitivity to growth signals and promotion of angiogenesis. A greatly increased understanding of the pathogenesis of the various underlying genetic and epigenetic lesions has accompanied the recent explosion of knowledge, coined the “omics” revolution. In a variety of cancer sites, we are already able to explain and classify much of the heterogeneity of tumour behaviour in terms of the underlying molecular lesions responsible. This capacity will increase with the scope of technologies becoming available, and being able to offer a corresponding tailored approach to therapy remains the ultimate goal. Keywords  Genetics • Epigenetics • Mutations • Carcinogenesis • Tumour suppressor genes • Oncogenes • DNA methylation • Histones

Introduction Despite the many innovations in cancer management, it is increasingly apparent that new advances in cancer therapy will rely on a greater understanding of treatment at a molecular level. A new “omics” language has developed with genomics, proteomics and metabolomics, all fields of translational research and terms used to bridge together traditional pure and applied science into the clinical setting. The clinical behaviour of HNSCC varies greatly from patient to patient, site to site and even within individual sub-sites, and its intrinsic heterogeneity in biological properties, which is common

R.J. Shaw (*) Head and Neck Surgery, Oral & Maxillofacial Surgery, University Hospital Aintree, University of Liverpool, Daulby Street, Liverpool L69 3GA, UK e-mail: [email protected]

in solid tumours, is reflected in the diversity of outcomes. These differences in tumour properties are a consequence of differing genetic and epigenetic changes as well as differing host responses. A better understanding of the genetic mechanisms of carcinogenesis will help to target therapies to specific characteristics of a patient’s tumour, central to the modern concept of personalised medicine. One example with clear and clinically exploitable differences in genetic mechanism is that of Human Papilloma Virus (HPV) mediated HNSCC, although this will not further discussed here as it is covered specifically in Chapter 10. Much of what follows is essentially a discussion of HPV negative HNSCC. Cancer results from the accumulation of molecular lesions which occur, and might be investigated, at the genetic, epigenetic, messenger RNA or protein level. The importance of genetic changes and frequency of the resultant disease have led to cancer being labelled the commonest human genetic disease. Often when we consider genetic diseases, we immediately think of inherited diseases. Fortunately inherited head and neck cancer syndromes are relatively uncommon, but there are several such entities predisposing to HNSCC which offer a valuable window on the events also critical to sporadic cancers. The great majority of sporadic cancers occur due to exposure to environmental mutagens. These mutagens cause genetic lesions that have a huge range of scale, from a single nucleotide to an entire chromosomal region being lost or gained. These genetic abnormalities occur more or less randomly rather than as an ordered sequence, and it is apparent that while some may be critical “drivers” to carcinogenesis, others are “bystander” events. Our ability to explore this disordered cancer genome is dependent on, and limited by, the availability of representative tumour models, high-quality tissue resource and the capacity of available technologies. Fortunately, there has been great progress in both areas in recent years and it is likely that this will significantly contribute to our understanding of cancer biology. Since the completion of the 15-year human genome project in 2003, the capability of the available “next-generation” sequencing techniques is such that an entire genome can be re-sequenced using a fraction of the resources previously required. It is now feasible to re-sequence individual tumours in attempt to

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_5, © Springer Science+Business Media, LLC 2011

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highlight which mutations appear critical to carcinogenesis. In many respects, DNA is an excellent resource for clinical biomarkers. Not only do many of the critical events occur at the DNA level, but also it is a highly stable macromolecule which is simply extracted and less prone to degradation and artefact than the more labile RNA and protein alternatives. One of the most consistent characteristics of cancer is genomic instability, resulting from loss of function in the DNA repair mechanisms. Each cell is daily subject to thousands of genetic insults which are generally repaired, or if beyond repair, the cell is destined for apoptosis. Inherited predisposition to cancer can either be those rare single gene autosomal recessive syndromes with a greatly increased risk (i.e. inherited cancer syndromes), or multiple polymorphisms more subtly affecting predisposition in the general population [i.e. single nucleotide polymorphisms (SNPs)]. In both cases, DNA damage and repair appear to be the common target. In exploring the results of comprehensive re-sequencing for sporadic human cancers, it has also become apparent that the commonest mutations seen are in also in the DNA repair machinery. In the last decade, interest has also grown in the epigenetics of cancer. The role of promoter hypermethylation has become a focus for research in many tumour sites, including HNSCC. Silencing of certain tumour suppressor genes (TSGs), central to the development of many solid tumours, may occur in the absence of genetic change, via aberrant methylation of CpG islands. Several promising avenues exist in attempting to translate this research field into the clinical management of HNSCC. The discussion in this chapter will present the genetic and epigenetic basis of HNSCC. The advances in molecular techniques which have contributed to our understanding of the development of head and neck cancer are also summarised.

Genetic Principles in Carcinogenesis Cancers arise from cells that have undergone heritable and non-heritable (somatic) genetic alterations which are then followed by clonal expansion. Theodore Boveri is generally credited as the father of this somatic mutation theory of carcinogenesis [1]. There is considerable evidence to support many of his predictions in his comprehensive theory on the origin of malignant tumours. These genetic alterations can involve activation of cancer promoting genes or inactivation of cancer suppressing genes. Multiple heritable changes are required for a normal cell to evolve into a cancer cell with evidence showing that this may involve between 3 and 10 genetic events, i.e. carcinogenesis is a multi-step and multifactorial process that involves multiple genes [2]. This is supported by histopathological observations revealing multiple stages of tumour progression from pre-malignancy to overt carcinoma, animal models of carcinogenesis and

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predisposition of cancer in individuals with heritable cancer syndromes. Mathematical models based on age-specific tumour incidence curves are consistent with 3–7 ­independent hits required for carcinogenesis [2]. These sequential accumulations of genetic abnormalities develop in a Darwinian fashion with those aberrations giving a selective advantage being propagated further.

Oncogenes and TSGs The two main types of genetic alterations in the development of cancer are activation of cancer promoting genes, otherwise known as oncogenes, which when activated support cell survival and proliferation, or inactivation of cancer suppressing genes, or TSGs, which when inactivated promote tumour development. Oncogenes are derived from alteration of cellular proto-oncogenes, a term used to describe genes which encode for proteins normally expressed that mediate positive cell growth or survival signals, i.e. an oncogene is an abnormally activated proto-oncogene. Oncogenes give cancer cells dominant gain of function with a selective growth advantage due to promoting uncontrolled cell proliferation. They can be activated by a number of mechanisms such as mutations that cause overexpression from gene amplification, increased transcription or by structural changes such as chromosomal translocation [3–5]. Most of the products of oncogenes override the normal cell cycle checkpoints allowing abnormal cell proliferation [6]. Oncogenes can be classified into one of the five broad functional groups [7]. They represent the differing stages at which their products are involved in the growth signal cascade, from the extracellular proteins such as growth factors and their transmembrane receptors (EGFR and erbB), the subsequent intracellular signalling transducer (ras and raf) to the eventual intra-nuclear transcription factors (c-myc). Other oncogene products include cell cycle regulators (cyclin D1) and inhibitors of apoptosis (bcl-2). The list of oncogenes associated with head and neck cancer is extensive. EGFR is commonly found to be overexpressed in HNSCC [8] and new treatments have arisen with cetuximab, a chimeric monoclonal antibody directed against EGFR. EGFR is a member of the ErbB protein family of cell surface receptors which works through the tyrosine kinase cascade. Downstream effects include activation of kinases and signal transducers [i.e. mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK) and the JaK/STAT pathway] which all result in activation of pathways involved in proliferation, apoptosis, invasion, angiogenesis and metastasis [9]. Genetic alterations in the EGFR pathway provide predictive biomarkers, as they correlate with response to therapy with cetuximab [10]. Interestingly, sensitivity to EGFR inhibition agents in other tumours has been shown to be accurately predicted by downstream RAS/RAF

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mutations in many tumour types [11]. Other oncogenes in HNSCC include D(delta)Np63-a(alpha) which belongs to the p53 family [12] and matrix metalloproteinases, which are involved in proliferation and migration [13]. TSG-encoded proteins have an inhibitory regulatory effect on growth, mediated via cell cycle, apoptosis, cell adhesion and DNA repair [14]. TSGs give cancer cells recessive loss of function and can be inactivated by genetic events such as mutation, deletions, or by epigenetic events such as DNA methylation or chromatin remodelling [15]. TSGs require both alleles to be altered before manifestation (i.e. homozygosity) unlike oncogenes, which can be activated by single allele activation (i.e. heterozygosity). This was first described by Knudson as the “double hit” theory for retinoblastomas [16, 17]. On inactivation of TSG cells lose their regulatory control leading to unchecked cell division and the development of cancer. p53, the “guardian of the genome,” was one of the earliest TSGs discovered in a broad range of cancers including head and neck cancer [18]. It is normally activated by stimuli that cause cellular stress such as radiation and carcinogenic toxins exposure. Such activation results in p53 regulating growth by influencing cell cycle control at the G1/S junction, a “checkpoint” in the cell cycle. Actively dividing cells pass such checkpoints to ensure that the progeny do not receive incomplete or damaged DNA. If the genetic impact of such stimuli is irreparable then it promotes apoptosis. p53 mutations are associated with a reduced survival in HNSCC patients with evidence for its role as a predictive biomarker [19–21]. Other important TSGs found in HNSCC include the CDKN2a locus which codes for p16 and p14 (ARF), discussed below. The DCC gene (deleted in colon cancer) on chromosome 18q21 is a conditional TSG [22] which mediates the growth effects by binding to netrin-1 and loss of the FAT gene [23] at 4p35 thought to play a role in cell–cell adhesion.

Fig. 5.1  The progression model for HNSCC. As genetic abnormalities (LOH and oncogene activation) accumulate over time, a corresponding histological progression is found. The rate of accumulation of these genetic changes is increased once genomic instability is established. The first stages of this progression axis may be heritable (susceptibility and predisposition disorders)

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The Multi-step Process of Carcinogenesis: The Genetic Progression Model The multi-step process of carcinogenesis reflects genetic alterations that drive the progressive transformation of normal cells to malignant cells and can be used to understand genetic events in terms of a timeline during the development of head and neck cancer. A genetic progression model in HNSCC has been postulated (Fig.  5.1) which demonstrate the common sequence of genetic losses [7, 24]. The frequency of genetic alterations in progressive lesions from dysplasia to carcinoma can correlate with the timing of such mutations along the progression axis. For example, there is a high frequency of loss of chromosomal material at loci 9p, 3p and 17p in dysplastic lesions indicating such events occur early in carcinogenesis. Loss of 13q and 8p are more ­frequently observed in carcinomas suggesting that they occur at later stages of carcinogenesis [24].

Early Events: Loss of 9p21 Region (CDKN2A Locus) The most common early loss found in HNSCC is ­chromosomal region 9p21 with 71% loss in pre-invasive and invasive lesions suggesting an early event [25]. This region encodes p16 and p14arf. The p16 gene encodes a cell cycle protein which inhibits cyclin-dependent kinases (CDK4 and CDK6); thereby preventing phosphorylation of Rb protein which would normally promote cell cycle progression from G1 to S phase [26–28]. p16 is frequently subject to epigenetic inactivation as well as loss.

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Loss of 3p Region Loss of chromosome 3p region is another early event in HNSCC found in oral dysplasia [29, 30]. The region includes the TSGs FHIT (fragile histidine triad gene) [37] and RSSFIA. RSSF1A may function in the effector molecule in the RAS-activated growth inhibition signalling pathway and is inactivated by hypermethylation and allelic loss [31].

Later Events: 17p Loss of Heterozygosity/p53 Mutation p53 is located on chromosome 17p13 as discussed earlier and its loss of function results in transformation as genetic abnormalities are allowed to persist unrepaired through the cell cycle and passing on to progeny. Mutations or deletions of p53 are associated with increased genomic instability which itself may hasten the rate and effect of further genetic mutations.

11q13 Amplification/Cyclin D1 Overexpression The oncogenes bcl-1, int-2, hst-1, EMS-1 and cyclin D1/ PRAD1 are implicated in the frequent amplifications (33%) at this site [32]. Cyclins are proteins involved in cell cycle regulation, the cyclin D1 gene product which activates Rb by phosphorylation, leading to cell cycle progression from G1 to S phases resulting in proliferation [33].

Fig. 5.2  Interaction of the hereditary conditions which predispose to HNSCC with the pathways of carcinogenesis (AT ataxia telangiectasia, BS Blooms syndrome, FA Fanconis anaemia, LFS Li-Fraumeni syndrome)

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There are many other examples of late genetic events in head and neck cancer including abnormalities detected at 3q26amplification, 13q21, 14q23, 4q21-q25 and 5q13 deletion [24].

Hereditary Conditions Predisposing to HNSCC Mutations may occur in the face of minimal exposure to carcinogens in a number of rare but mechanistically interesting inherited cancer syndromes (Table  5.1). During somatic mitosis, errors are observed in DNA replication. A failure in the regulatory cellular processes involved in correcting these replication errors contributes to carcinogenesis since accurate replication and repair of DNA is essential to genomic integrity. The regulatory processes involve systems to detect the abnormal DNA and mechanisms to repair them, which, if not possible, will lead to programmed cell death (or apoptosis) [34]. Defects in any part of this chain of events of recognition, repair or apoptosis result in unresolved genomic instability and can be demonstrated in hereditary conditions with a predisposition to cancer (Fig. 5.2). Table 5.1  Hereditary conditions predisposing to HNSCC Fanconis anaemia Blooms syndrome Ataxia telangiectasia Xeroderma pigmentosum Li-fraumeni syndrome Lynch II syndrome

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Fanconi Anaemia Fanconi anaemia (FA) is a rare autosomal recessive disorder (incidence 1:350,000) that was first described in 1927 [35]. It is characterised by various congenital malformations, progressive bone marrow failure and tumour development [36]. The disease involves many organs and patients typically present with hyper-pigmentation, skeletal anomalies, growth retardation, learning disability and risk of secondary malignancies including HNSCC at a young age. The genes associated with FA have a caretaker role in the protection against carcinogenesis, i.e. they maintain the integrity of the genome by DNA repair mechanisms. FA is defined by its cellular hypersensitivity to DNA cross-linking agents and two genetic defects have been suggested that determine the development of cancer, defective chromosomal stability and immunodeficiency [37]. The genes associated with the FA including BRCA1, BRCA2 (also mutated in familial breast cancers), FANCD2 and FANCG are frequently also affected in sporadic HNSCC. This suggests that the process leading to early occurrence of oral cancer in FA patients follows a similar pathway of carcinogenesis to non-FA sporadic cancer patients, particularly in younger patients [38]. This is supported by differences in expression levels of Fanconi-related genes in OSCC of younger compared to older patients. It leads to the conclusion that sporadic tumours in younger patients also occur through defective carcinogen metabolism or DNA repair mechanisms (see below).

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a super complex of BRCA1-associated proteins named BASC (BRCA1-Associated genome Surveillance Complex). This surveillance complex includes proteins involved in replication repair processes found in ataxia telangiectasia (ATM), hereditary non-polyposis colorectal carcinoma (HNPCC) (MLH1 and MSH2) [39] and some of the Fanconi complementation group of proteins (FANCA, FANCG, etc.) [40]. There are also interactions between the BLM protein and many other proteins including p53 [41].

Ataxia Telangiectasia Ataxia telangiectasia (AT) is a debilitating and progressive neurodegenerative disease of childhood and characteristic defects include neurodegeneration, immune dysfunction, radiosensitivity and cancer predisposition. The underlying cause of the disease is mutation in the ATM (ataxia telangiectasia, mutated) gene located on 11q22-23 [42], a common deletion site in HNSCC [43]. Its product is a protein kinase involved in the cellular response to DNA damage [44]. ATM is also involved in immune system maturation and meiosis. Cancer predisposition is mainly of the lymphoreticular ­system and is also linked to head and neck malignancy in younger patients [45]. The ATM gene is involved in surveillance of DNA damage, activation of repair enzymes or apoptosis if such damage is irreparable. After DNA damage, it undergoes autophosphorylation initiating a signalling ­cascade involving cell cycle regulators including p53, BRCA1, p53-binding protein 1 (p53BP1) and the checkpoint kinase CHK2 [46].

Blooms Syndrome Bloom’s syndrome (BS) is also an autosomal recessive disorder associated with Ashkenazi Jewish ancestry belonging to the “chromosomal breakage syndromes.” It is characterised by marked genetic instability and is associated with a greatly increased risk of a wide range of cancers including head and neck malignancy. Features include growth retardation, hypersensitivity to sunlight, facial sun-sensitive telangiectactic erythema, skin pigmentation in non-sun-exposed skin and moderate to severe immunodeficiency. Patients are predisposed to develop either haematological or solid tumours. BS is characterised by a high level of sister chromatid exchanges, when two chromatids form during late prophase of mitosis, break and rejoin with one another and thereby physically switching positions on the chromosome. This increased frequency of chromosomal breakages or interchanges occurring spontaneously or following exposure to DNA damaging agents. The genetic instability arises through mutations in both copies of the BLM gene, located on chromosome 15q26. The BLM protein interacts with ­proteins involved in genomic maintenance and stability and

Xeroderma Pigmentosum Xeroderma pigmentosum (XP) is an autosomal recessive ­disease characterised by a severe predisposition to ultraviolet light-induced skin cancers. There are two major clinical forms, one involving progressive degenerative changes of the skin and eyes and the other also includes progressive neurological degeneration [47]. There is an extreme sensitivity to sunlight with cutaneous symptoms ranging from sunburn to overt carcinoma. The risk of squamous cell carcinoma is elevated by 100,000-fold with a median onset of skin cancer at 8 years of age. XP is the archetype of the family of nucleotide excision repair (NER) diseases [48] deficient in a gene product required in the excision of damaged DNA. The NER excises damaged single strands of DNA and replace them with a new sequence of bases using a template for base pairing the intact strand of DNA opposite the damaged site. Abnormalities in this repair system leads to high levels of specific modifications of crucial regulatory genes in skin cells leading to cancer as well as resulting in chromosomal breakage [48].

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Li-Fraumeni Syndrome Li-Fraumeni syndrome is a rare autosomal dominant syndrome characterised by a predisposition to cancers including sarcomas, leukaemia’s and brain tumours. The early onset malignancy (<45 years), usually a sarcoma, is associated with a family history of multiple cancers. There is frequently a germline mutation of the TSG p53 [49] and other TSGs such as CHEK2. They have a 90% lifetime risk of developing cancer [50] and are at risk of developing both lung and laryngeal carcinomas.

Lynch Syndrome II HNPCC is an autosomal dominant inherited familial cancer syndrome in which patients are susceptible to colorectal cancer without diffuse polyposis. Lynch Syndrome II has additional features to Lynch I and has an association with extracolonic cancers including laryngeal cancer [51]. These patients have a defect in the DNA mismatch repair enzyme hMSH2, an important enzyme in genetic instability and carcinogenesis of HNPCC [52].

Genetic Predisposition and Mutagen Sensitivity Inherited chromosomal instability syndromes represent one end of a spectrum of DNA repair defects. There are individual variations in the efficiency of DNA repair systems within “normal” individuals who subsequently have an increased tendency for DNA damage from carcinogen exposure consequently increasing their susceptibility to cancers [53]. Environmental exposure to carcinogens implicated in head and neck carcinogenesis cannot fully account for the development of cancer alone since not all heavy smokers develop cancer and likewise not all cancer patients are exposed to alcohol or tobacco [53–55]. Individuals with an inherited genetic susceptibility to head and neck cancer can have defects in systems used to maintain DNA after exposure to such carcinogens [54]. Mutagen sensitivity and polymorphisms in DNA repair enzymes or carcinogen metabolising enzymes support the role of hereditary susceptibility for head and neck cancer and giving some explanation to the variable risk from carcinogen exposure. Studies have demonstrated odds ratios of 1.5–2.5 for cancer development in those predisposed by mutagen sensitivity polymorphisms. Greater predictive power is found when combining mutagen sensitivity tests with other risk factors

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such as alcohol and smoking in head and neck cancer. Both epidemiological and ­molecular evidence support the role of heritable genetic susceptibility for head and neck cancer. Familial aggregation studies have demonstrated that family history is a significant risk factor for head and neck cancer [56]. Genetic polymorphisms in carcinogen metabolising enzymes (xenobiotic metabolising enzymes) frequently occur in the population, partially explaining this susceptibility. Both phase 1 and phase 2 enzymes have been implicated. Phase 1 enzymes include the cytochrome P450 enzymes CYP1A1, CYP1B1, CYP2D6 and CYP2E1. Phase II enzymes include Glutathione S-transferase enzymes GSTM1, GSTT1 and GSTP1. Metabolic polymorphisms such as those in the cytochrome P450 (CYP)1A1 gene are associated with oral cancer risks in some populations with cancer resulting from relatively lower smoking exposure than those with differing genotypes [57, 58]. A meta-analysis assessing the associations of oral cancer risk with CYP1A1 genetic variation suggested that it might not be a risk factor, whereas the GSTM1 null genotype significantly increased susceptibility to oral cancer in Asians but not in Caucasians [59]. Other polymorphisms have been found to be protective, e.g. GSTT1 null genotype among Indian tobacco users [60].

Techniques Used to Detect Genetic Changes in Cancer See Table 5.2.

Cytogenetics Cytogenetics, the study of chromosomal rearrangements, was one of the earlier methods of determining the site of genetic abnormalities related to the pathogenesis of cancer. With karyotype analysis, cultured cells are arrested in ­metaphase and a visual analysis made of gross genetic changes such as translocations, breakpoints and areas of gain

Table 5.2  Techniques used to detect genetic changes in cancer Cytogenetics Comparative/genomic hybridisation (CGH) Fluorescent in situ hybridisation (FISH) Microsatellite alterations DNA microarray techniques SNP arrays Sanger sequencing Next-generation sequencing

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or loss. At metaphase, the chromosomes appear as long arms (p) and short arms (q) joined at the centromere. The ­disadvantage of such gross techniques is that alterations at a DNA base level are missed and abnormalities found are only a snapshot in time. The commonest areas of gain are on the short arm of chromosomes 1, 3, 8 and 15. The commonest areas of loss are on the long arms of chromosomes 8, 13, 14, 15 and 11q. Cytogenetics can be used as a screening technique to identify gross losses or gains but subsequent identification of specific genes requires functional studies to determine gene properties.

Fluorescent In Situ Hybridisation and Comparative Genomic Hybridisation Techniques have developed over time to improve the resolution of karyotype analysis and molecular cytogenetic technology such as fluorescent in situ hybridisation (FISH) and comparative genomic hybridisation (CGH) have increased the ability to identify genetic alterations in cancer cells. These higher resolution techniques have found area of loss commonly in 1p, 3p, 4p, 5q, 8p, 10p, 11q, 13q and 18q and areas of gain at 1q, 3q, 5p, 7q, 8q, 9q, 11q, 12p, 14q and 15q [61, 62]. FISH is a technique in which sequence-specific fluorescent probes are detected after hybridisation using microscopy detecting gains (amplifications) or losses (deletions). It is an efficient and a reproducible approach for precise localisation of specific sequences and allows visualisation of copy number per cell. CGH is a fluorescence in situ technique developed to look at chromosome losses or gains which are unbalanced throughout the genome. It allows mapping of chromosome imbalances using total genomic DNA as a probe. In this technique, normal metaphase ­chromosomes are hybridised with differentially fluorescence labelled DNAs (tumour with green and normal with red) and fluorescence in different regions measured for increased or decreased intensity which would measure over or under expressed areas of tumour DNA. While cytogenetics have provided an insight into specific regions of tumour DNA, CGH enables comparisons of entire genomes of malignant and normal cells. It also allows retrospective ­studies with archival tissues overcoming the difficulties of conventional cytogenetic analysis of solid tumours. The most frequently observed changes are copy number changes on chromosomes 3 and 5. Examples of TSGs found at these locations include VHL on 3p [63] and FAP on 5q [33, 64]. These studies also identified the region 11q13 in a HNSCC with amplification of this region correlates with aggressive growth and a late event in the progression axis [65]. CGH arrays have been used in profiling HNSCC revealing ­previously unknown oncogenes as well as ­predicting ­chemoradiosensitivity [38].

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DNA Microarray Techniques Edwin Southern, a British biologist, first described in 1975 the use of labelled nucleic acids in a known DNA sequence (or a probe) to identify complementary DNA fragments through hybridisation of base pairs, a process known as Southern Blotting [66]. The DNA probes used were labelled with a radioisotope or a fluorescent tag. The methods of probe detection for DNA have been miniaturised into microarray techniques which enable detection of several thousands of DNA or RNA sequences at a time. The process is the reverse of Southern Blotting with the probe being placed on an immobile surface such as glass, silicone or nylon and exposed to free nucleic acid to be analysed [67]. It has developed CGH to several orders of magnitude hybridising normal and cancer DNA to several millions of probe sequences and allowing for a much finer map of genetic abnormalities [38]. This technology has been used to identify specific regions of gain such as 8q22 and the LRP12 gene [29].

SNP Arrays Work on the human genome project led to the discovery of SNPs, scattered areas of altered DNA sequences which ultimately have no impact on protein expression and so no adverse effect in normal individuals. Since these SNPs cluster in populations they can be used as identification as well as markers for genetic predisposition if located in DNA repair genes or detoxifying enzymes for environmental toxins and can indirectly provide information on gain or loss of specific genomic regions [61].

Sequencing and Next-Generation Sequencing Genomic sequencing has greatly progressed since dideoxy chain termination sequencing in 1970s [68]. The automated sequencer accelerated the process and subsequent development of the “shotgun” technique saved more time by splitting the genome into smaller segments. In the past few years, another generation of sequencers have been launched that can read as much DNA in a day that would previously taken years. Next-generation sequencing is a technique central to the emerging field of personalised medicine and produce large arrays of templates with each one read simultaneously so millions of templates can be read at once with thousands of times the throughput of Sanger’s technique. With the everreducing costs of the technology, there is the real prospect of developing biomarkers based on genome-wide mutational screens looking at patterns of mutations or genomic landscapes to stratify the risk of cancer rather than looking at individual genetic mutations.

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The Epigenetics of Head and Neck Cancer: Epigenetics: The Role of DNA Methylation, Histone Modifications and the Nucleosome As each cell in the body contains the same DNA code, but the morphology and behaviour of these cells differs greatly, it can readily be appreciated that much of this variation arises from the way that the DNA is interpreted. This change in interpretation is known as epigenetics, providing an extra layer of processing in addition to the basic genetic paradigm of DNA → RNA → protein proposed by Crick and Watson. Epigenetic changes are heritable modifications of DNA with information content that influence phenotype, but that are not associated with changes in nucleotide sequence. The regulation of gene expression is by the controlling influence of the proteins surrounding the DNA molecules known as histones. Chemical modifications of both histones (the “histone code”) and DNA (the “DNA methylome”), control the availability of genes for transcription, and in turn has a fundamental influence on cell differentiation. It is now beyond doubt that epigenetic dysregulation has a major part to play in carcinogenesis; however, before attempting to understand the changes seen in cancer, it is important to review the normal physiological role of epigenetics. The most important aspects of epigenetic regulation are methylation of gene promoters and modification of histone proteins. Gene promoter regions are particularly interesting stretches of DNA usually leading up to the transcriptional start sites. From the four nucleotides seen in DNA (A, C, T and G), it might readily be assumed that the frequency of any particular dinucleotide combination in any particular stretch of DNA might be approximately 1/16th. However, one ­particular combination, CG, has a very characteristic pattern.

Fig. 5.3  Effect of gene promoter methylation on gene expression

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For much of the genome, the CG dinucleotide has been evolved out because it is highly mutation-prone. In contrast, the CG content within certain defined stretches of DNA is particularly high, upwards of 50%, and these regions are called CpG islands (the “p” merely refers to the phosphordiester bond seen between all nucleotides in DNA). Definitions of CpG islands vary, but typically refer to stretches of around 200 base pairs with >50% CpG content upstream of mammalian genes. Chemical modification of the cytosine by methylation tends to occur in concert along the gene promoter. This is associated with transcriptional silencing via conformational changes in the surrounding histones (Fig. 5.3). The “default” CpG methylation pattern of humans is in two different states depending on the location. For scattered CpG not associated with gene promoters, almost all are methylated. Within CpG islands, most are normally unmethylated. Changes in methylation are brought about by a balance of two coordinating influences: first waves of de novo methylation [69] sweeping the genome and second active “resetting” demethylation, probably mediated through histone modifications [70]. This DNA methylome is reset early in embryogenesis and then re-established around the time of implantation. Further targeted alterations subsequently occur throughout differentiation and are associated with the loss of pluripotential state. The embryonic stem cell is principally characterised by its epigenetic signature. The interplay between chromatin and DNA methylation is somewhat complex and currently the focus of much research. It appears that in some circumstances, the DNA methylation pattern creates changes in histones, but in ­others the reverse relationship is found. In a highly simplified model of transcriptional regulation, unmethylated DNA is found in association with acetylated histones with a relaxed chromatin

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Fig. 5.4  Methylation and histone acetylation co-ordinate transcriptional availability. HAT histone acetyltransferase, HDAC histone deacetylase, m methylated CpG dinucleotide

structure amenable to transcription. In methylated DNA, histone deacetylase is recruited creating a much tighter conformation of deacetylated histones which are not available for transcription and hence the gene is silenced (Fig. 5.4). A pair of each of the core histones (H2A, H2B, H3 and H4) make up an octomer in which DNA is wound around to from a nucleosome. The N-terminal tails of histones are subject to a wide variety of covalent modifications such as acetylation, methylation and phosphorylation, which have influence over conformation and subsequent gene expression. Heterochromatin is the term used to describe highly packed regions which are transcriptionally silenced. Methylation of histone H3 at lysine 9 (H3K9) and 27 (H3K27) are important modifications in heterochromatin. Gene repression induced by histone methylation and heterochromatin formation is readily reversible and common in cellular differentiation. Gene repression induced by DNA methylation leads to permanent gene silencing and is seen in various physiological and pathological processes such as imprinting and cancer [71]. It is likely that the DNA methylation pattern of a cell might be the template used to reconstitute the epigenetic programme following cell division. A third epigenetic mark worthy of consideration is nucleosome occupancy. Nucleosomes are known to be dynamic structures and tend to be depleted in promoter regions [72]. Removal of nucleosomes correlates with transcriptional activation, and often this can be related to only one or two nucleosomes near the transcriptional start site [73]. Presumably, this is because transcription binding sites become more accessible.

Epigenetic Drivers of Carcinogenesis: Challenging the Genetic Paradigm It has become increasingly apparent that cancer is as much a disease of misdirected epigenetics as genetic mutations and losses. As an illustration, recent results suggest that as many as 5% of known gene promoters (i.e. 1,500–2,000 genes) are methylated in a typical solid tumour [74] compared with, typically, 11 gene mutations [75]. While it is now beyond dispute that silencing of many TSGs important to cancer occurs through DNA methylation, the models available to explain an overall contribution of epigenetics to cancer have evolved with the technologies available to study them. Initially, it was understood that the principal change in cancer was hypomethylation of genome-wide CpGs [76], but eventually it was realised that the functional significance of promoter methylation of a smaller number of TSGs [77] might have greater functional relevance. One model is that methylation patterns arise through a process of selection. Some evidence suggests that tumours have upregulation of DNA methyltransferase enzymes and that those cells gaining a growth advantage from the clones of cells primed for malignant transformation [78]. These epigenetic events may then occur in pre-invasive lesions, involving disruption or over-activation of key developmental pathways and cellsignalling properties. This, so called, “epigenetic addiction” [79] may then predispose to later genetic mutations and genomic instability ultimately causing malignant transformation. In this model, one may hypothesise that epigenetic

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events may be the most valuable in predictive modelling in potentially malignant H&N lesions. Advances in genomewide methylation technologies have, however, revealed that not all epigenetic events appear to occur in the TSGs expected. It is seen that many methylation events occur in gene promoters not previously implicated in cancer and with no obvious mechanistic links. An alternative hypothesis is that certain genes are earmarked for methylation and it has been shown that genes frequently methylated in cancer have specific tri-methylation of lysine 27 in histone H3 in their nucleosomes [80]. This suggests that cancer targeted de novo methylation may be programmed by a pre-ordained epigenetic code that physiologically has a role in marking embryonic genes for repression. Many hypermethylated genes in adult cancers are Polycomb group marked (H3K27 me) in embryonic stem cells and there are many, and unexpected, similarities in higher-order chromatin conformation between stem cells and adult cancers [81].

Molecular Assay of Epigenetic Alterations in Cancer As the technological platforms which support detection of methylation have generally lagged behind other genomic methods, the translational potential of the DNA methylome has remained relatively unexplored. Much of the previous HNSCC literature has concentrated on a known published cohort of methylated genes [82, 83], clearly limiting progress. Methylation assays have previously relied upon methylation-sensitive restriction enzymes or methylcytosine antibodies which greatly limit the number of samples analysed and precision of resulting data. Probably, the largest breakthrough in methylation assays was bisulphite conversion, in which the methylation code is converted to a C/T polymorphism. This then allows the creation of methylationspecific PCRs [84] and a similar process might be made semi-quantitative by using real-time PCR [85]. Methylationspecific PCR can be criticised as being only as specific as the primers and conditions allow, and without doubt some of the applications previously suggested push the envelope of reliable and reproducible performance of PCR. It must also be appreciated that the loss of sequence complexity accompanying bisulphite conversion also predicates towards the loss of specificity. Methods previously described for SNiP analysis might then be “borrowed” for methylation assays, such as pyrosequencing [86–88]. Alternative methods use standard Sanger sequencing of bisulphite-converted DNA, but the impact of massive parallel sequencing has yet to be realised in epigenetics. Various multiplexed and array methods have been made available, either relying on modifications of SNiP arrays [89] for use with bisulphite conversion or using

R.J. Shaw and J. Dhanda

r­ estriction enzymes. In addition, more indirect methods have been improvised to detect potential methylation targets using pharmacological unmasking. It is possible to shortlist those genes upregulated by the use of demethylating agents in cancer cell lines using expression arrays [90, 91]. In order to detect histone modifications, where methods of chromatin immunoprecipitation predominate, effective scaled-up assays are not currently available.

Clinical Application of Epigenetics in Head and Neck Cancer A comprehensive list of genes subject to promoter methylation in HNSCC is not yet available, and the previously published candidates [82, 83] are subject to regular additions. The genes and pathways subject to methylation are spread across the broad range of cellular functions. It may be that some of the methylation events seen as being relatively frequent in HNSCC are not critical mechanistic determinants of progression; however, others such as promoter methylation of p16 (CDKN2) [87, 92] appear to be highly likely to have functional relevance. Whether this is of importance to a suggested clinical application is highly dependent on the intended use of the assay and this brushes on broader issues concerning on molecular biomarkers are discussed below. Studies in ­several tumour sites (in particular, colorectal cancer [93]) highlight the significance of the CpG island methylation phenotype (CIMP), with distinct features of histology, biological aggression and outcome. A cluster of tumours with a greater degree of promoter methylation than would be predicted by chance alone are designated CIMP+ve. In HNSCC, initial studies suggest that this group had less aggressive tumour biology and excite a greater host inflammatory response [94]. The exact mechanisms underlying CIMP remain obscure, one may now speculate that the affected genes are histone H3K27 targets for EZH2-containing Polycomb complex [80].

Epigenetic Biomarkers in HNSCC The intended use of biomarkers can vary quite dramatically between assays. Biomarker research can resemble an uncertain navigation of the minefield between the discovery of an interesting observation in the research lab and the adoption of a proven biomarker for the benefit of patients in the clinic. Predictive biomarkers are designed to help make treatment decisions as they predict for response to ­certain treatments, whereas prognostic biomarkers give an indication of the likely outcome for survival. A further class of biomarkers predicts for the mere presence of disease in

5  Genetics and Epigenetics of Head and Neck Cancer

surrogate ­specimens such as saliva, blood or surgical margins. In many regards, DNA methylation appears to be a very promising aberration for biomarker applications as it can be detected with great specificity and sensitivity in many biological specimens [95]. Further, it may be present in a relatively high proportion of tumours encountered (e.g. cyclin A1 methylation in 50% of HNSCC [87, 91]), which is in contrast to specific p53 mutations which are, individually, relatively uncommon. Although mutations are irreversible events, there is the possibility of pharmacological reversal of methylation and histone changes. The ability to detect tumour-specific methylation events with great sensitivity results from methylation-specific assays which can detect methylated DNA, certainly in 1:1,000 concentrations [85], and maybe in much lower proportions [96]. It is possible then, at least in theory, to screen populations for cancerspecific events in saliva, perhaps using panels of methylation events [97] or to offer surveillance to post-treatment patients using their previous cancer’s epigenetic fingerprint [98]. It is also possible to search for tumour-specific methylation in histologically negative resection margins in order to optimise adjuvant treatment [99–101]. Management of the patient with dysplasia of the head and neck depends on accurate prediction of transformation and pathological grading fails in some regards. Bringing together the promise of epigenetic biomarkers within non-invasive sampling and also the mechanistic relevance of epigenetics in priming the molecular field for further genetic events seems logical in this setting. It has now been established that 3p and 9p losses represent very effective genetic biomarkers of progression within H&N dysplastic lesions [24]. Early evidence suggest that methylation may have some promise in this field [102] and one longitudinal study has found p16 methylation to be a specific predictor of malignant progression in oral dysplasia [103]. It may be possible to make a prognostic evaluation of a tumour prior to definitive treatment by epigenetic analysis of the biopsy. In this way, the patient’s treatment may be individualised by methylotype. The full impact of genome-wide methylation profiling and its prognostic value has yet to be evaluated. Realising the potential in order for any of these applications to be clinic-ready is self-evidently a painstaking process.

Epigenetically Directed Therapy in HNSCC Several suggestions have been made that promoter methylation of specific genes may indicate a particular tumour’s sensitivity to a drug. Such studies have yet to make an impact in the treatment of HNSCC. In the treatment of gliomas, it has been found that MGMT promoter methylation is a useful predictor of the responsiveness to alkylating agents such as Temozolomide [104]. Perhaps of more relevance is

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that CHFR methylation predisposes cancer to increased ­sensitivity to taxanes [105] which may be a useful line of investigation as TPF-induction chemotherapy gains ground in HNSCC. Epigenetic alterations are particularly interesting as characteristics of cancer as they can potentially be reversed in drug treatment. A great number of epigenetically directed drugs are now entering clinical trials, particularly in the field of haematological malignancy. Demethylation agents such as 5-azacytidine and decitabine are now licenced for the treatment of myelodysplasia. The use of histone deacetylase inhibitors as single agents has limited usefulness but shows promise in combination with demethylating agents. The emergence of drug-resistant clones accompanying loss of DNA mismatch repair with MLH1 hypermethylation has been identified as a frequent issue in treatment failure in ovarian cancer. At least in vitro, this has been addressed by combination therapy with both demethylating agents and standard chemotherapy [106]. Clearly this field is in its infancy, and provided epigenetic therapy can be administered with acceptable toxicity; methylation assays are readily available to demonstrate adequate pharmacodynamic effects.

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105 DNA methyltransferase revealing nucleosome positions at a single molecule level. Nucleic Acids Res. 2005;33(20):e176. 74. Schuebel KE, Chen W, Cope L, Glockner SC, Suzuki H, Yi JM, et al. Comparing the DNA hypermethylome with gene mutations in human colorectal cancer. PLoS Genet. 2007;3(9):1709–23. 75. Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, et al. The consensus coding sequences of human breast and colorectal cancers. Science. 2006;314(5797):268–74. 76. Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983;301(5895):89–92. 77. Sakai T, Toguchida J, Ohtani N, Yandell DW, Rapaport JM, Dryja TP. Allele-specific hypermethylation of the retinoblastoma tumorsuppressor gene. Am J Hum Genet. 1991;48(5):880–8. 78. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128(4):683–92. 79. Baylin SB, Ohm JE. Epigenetic gene silencing in cancer – a mechanism for early oncogenic pathway addiction? Nat Rev Cancer. 2006;6(2):107–16. 80. Schlesinger Y, Straussman R, Keshet I, Farkash S, Hecht M, Zimmerman J, et al. Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer. Nat Genet. 2007;39(2):232–6. 81. Tiwari VK, McGarvey KM, Licchesi JD, Ohm JE, Herman JG, Schubeler D, et al. PcG proteins, DNA methylation, and gene repression by chromatin looping. PLoS Biol. 2008;6(12):2911–27. 82. Ha PK, Califano JA. Promoter methylation and inactivation of tumour-suppressor genes in oral squamous-cell carcinoma. Lancet Oncol. 2006;7(1):77–82. 83. Shaw R. The epigenetics of oral cancer. Int J Oral Maxillofac Surg. 2006;35(2):101–8. 84. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation ­status of CpG islands. Proc Natl Acad Sci USA. 1996;93(18): 9821–6. 85. Easds CA, Danenberg KD, Kawakami K, et al. Methylight: a highthroughput assay to measure DNA methylation. Nucleic Acids Res. 2000;28:E32. 86. Colella S, Shen L, Baggerly KA, Issa JP, Krahe R. Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites. Biotechniques. 2003;35(1):146–50. 87. Shaw RJ, Liloglou T, Rogers SN, Brown JS, Vaughan ED, Lowe D, et al. Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br J Cancer. 2006;94(4):561–8. 88. Shaw RJ, Hall GL, Lowe D, Liloglou T, Field JK, Sloan P, et al. The role of pyrosequencing in head and neck cancer epigenetics: correlation of quantitative methylation data with gene expression. Arch Otolaryngol Head Neck Surg. 2008;134(3):251–6. 89. Bibikova M, Lin Z, Zhou L, Chudin E, Garcia EW, Wu B, et al. High-throughput DNA methylation profiling using universal bead arrays. Genome Res. 2006;16(3):383–93. 90. Hoque MO, Kim MS, Ostrow KL, Liu J, Wisman GB, Park HL, et  al. Genome-wide promoter analysis uncovers portions of the cancer methylome. Cancer Res. 2008;68(8):2661–70. 91. Tokumaru Y, Yamashita K, Osada M, Nomoto S, Sun DI, Xiao Y, et al. Inverse correlation between cyclin A1 hypermethylation and p53 mutation in head and neck cancer identified by reversal of epigenetic silencing. Cancer Res. 2004;64(17):5982–7. 92. El-Naggar AK, Lai S, Clayman G, Lee JK, Luna MA, Goepfert H, et al. Methylation, a major mechanism of p16/CDKN2 gene inactivation in head and neck squamous carcinoma. Am J Pathol. 1997;151(6):1767–74. 93. Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP. CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci USA. 1999;96(15):8681–6.

106 94. Shaw RJ, Hall GL, Lowe D, Bowers NL, Liloglou T, Field JK, et al. CpG island methylation phenotype (CIMP) in oral cancer: associated with a marked inflammatory response and less aggressive tumour biology. Oral Oncol. 2007;43(9):878–86. 95. Laird PW. The power and the promise of DNA methylation markers. Nat Rev Cancer. 2003;3(4):253–66. 96. Shaw RJ, Akufo-Tetteh EK, Risk JM, Field JK, Liloglou T. Methylation enrichment pyrosequencing: combining the specificity of MSP with validation by pyrosequencing. Nucleic Acids Res. 2006;34(11):e78. 97. Righini CA, de Fraipont F, Timsit JF, Faure C, Brambilla E, Reyt E, et al. Tumor-specific methylation in saliva: a promising biomarker for early detection of head and neck cancer recurrence. Clin Cancer Res. 2007;13(4):1179–85. 98. Carvalho AL, Jeronimo C, Kim MM, Henrique R, Zhang Z, Hoque MO, et al. Evaluation of promoter hypermethylation detection in body fluids as a screening/diagnosis tool for head and neck squamous cell carcinoma. Clin Cancer Res. 2008;14(1):97–107. 99. Goldenberg D, Harden S, Masayesva BG, Ha P, Benoit N, Westra WH, et al. Intraoperative molecular margin analysis in head and neck cancer. Arch Otolaryngol Head Neck Surg. 2004;130(1):39–44. 100. Shaw RJ, Hall GL, Woolgar JA, Lowe D, Rogers SN, Field JK, et al. Quantitative methylation analysis of resection margins and lymph nodes in oral squamous cell carcinoma. Br J Oral Maxillofac Surg. 2007;45(8):617–22.

R.J. Shaw and J. Dhanda 101. Tan HK, Saulnier P, Auperin A, Lacroix L, Casiraghi O, Janot F, et al. Quantitative methylation analyses of resection margins predict local recurrences and disease-specific deaths in patients with head and neck squamous cell carcinomas. Br J Cancer. 2008; 99(2):357–63. 102. Lopez M, Aguirre JM, Cuevas N, Anzola M, Videgain J, Aguirregaviria J, et  al. Gene promoter hypermethylation in oral rinses of leukoplakia patients – a diagnostic and/or prognostic tool? Eur J Cancer. 2003;39(16):2306–9. 103. Hall GL, Shaw RJ, Field EA, Rogers SN, Sutton DN, Woolgar JA, et al. p16 Promoter methylation is a potential predictor of malignant transformation in oral epithelial dysplasia. Cancer Epidemiol Biomarkers Prev. 2008;17(8):2174–9. 104. Esteller M, Garcia-Foncillas J, Andion E, Goodman SN, Hidalgo OF, Vanaclocha V, et  al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med. 2000;343(19):1350–4. 105. Banno K, Yanokura M, Kawaguchi M, Kuwabara Y, Akiyoshi J, Kobayashi Y, et al. Epigenetic inactivation of the CHFR gene in cervical cancer contributes to sensitivity to taxanes. Int J Oncol. 2007;31(4):713–20. 106. Steele N, Finn P, Brown R, Plumb JA. Combined inhibition of DNA methylation and histone acetylation enhances gene ­re-expression and drug sensitivity in  vivo. Br J Cancer. 2009; 100(5):758–63.

Chapter 6

Immunology of Head and Neck Cancer Steve C. Lee and Robert L. Ferris

Abstract  The immune system plays a key role in the ­progression of head and neck cancer. A greater understanding of the important contribution of the dysregulation and evasion of the immune system in the development and evolution of head and neck cancers should lead to improved therapies and outcomes for patients. Head and neck cancer evades the host immune system through manipulation of its own immunogenicity, production of immunosuppressive molecules, and promotion of immunomodulatory cell types. Also, the immune system can be exploited to promote metastasis, angiogenesis, and growth. In this chapter, we review basic immunology as it relates to head and neck cancer and discuss the theory of cancer immunosurveillance and immune escape. Current research on cytokines as biomarkers, cancer stem cell tumor antigens, and immunotherapeutic strategies are presented. Keywords  Immunology • Immunotherapy • Head and neck cancer • Biomarkers • Immune evasion • Immune surveillance • Monoclonal antibodies

Introduction The immune system plays a key role in the progression of head and neck cancer. A greater understanding of the important contribution of the dysregulation and evasion of the immune system in the development and evolution of head and neck cancers should lead to improved therapies and outcomes for patients. In this chapter, we review basic immunology as it relates to head and neck cancer and discuss the theory of cancer immunosurveillance and immune escape. There has been a recent renaissance in the idea that nascent cancer cells are destroyed by the immune system before tumor formation can occur (termed immune ­surveillance). R.L. Ferris (*) University of Pittsburgh Cancer Institute, 200 Lothrop Street, Suite 500, Pittsburgh, PA 15213 e-mail: [email protected]

Derangements in the immune system or alterations in the transformed cells may allow immune escape that allows the cancer to become manifest. Once tumor is established, there are a myriad ways in which it interacts with the immune system. Transcription factors such as NFk(kappa)B (nuclear factor kappa-light-chain-enhancer of activated B cells) and STAT3 (signal transducers and activators of transcription), which are usually dysregulated in tumor-promoting inflammatory states in response to cytokine stimuli, are aberrantly activated in tumor cells and are intensively studied as possible targets for therapeutic intervention. Tumors themselves produce cytokines such as TGF-b(beta), IL-6, and IL-10, which suppress cell-mediated antitumor immunity. In response to inflammatory stimuli, head and neck cancer cells also can express receptors which are involved in lymphocyte and dendritic cell migration. Expression of these receptors by tumor cells, such as CCR7 and CXCR4, constitute immune exploitation of established signals intended for immune cells and have been associated with tumor invasion, metastasis, and cell survival, leading to treatment resistance. Another recently espoused theory is the idea that tumors are comprised of a heterogenous cell population in the tumor microenvironment that includes a special subpopulation of cancer stem cells (CSC) that are able to recreate the entire tumor phenotype and potentially evade immune recognition. These cells appear to be more resistant to conventional chemotherapy and radiation, and may not possess the same tumor antigen expression or T-cell recognition as non-CSC. In head and neck cancer patients, there appear to be global alterations in the functional state of the immune system, as evidenced by changes in serum cytokines, chemokines and other immune-related biomarkers in cancer patients. There is considerable investigation focusing on the identification of serum biomarkers to monitor cancer progression, prognosis, treatment response, and relapse. Finally, we describe various immunotherapeutic strategies designed to utilize the immune system to stimulate elimination of cancer. These include ­cancer vaccines using tumor peptide antigens or viral, bacterial, and DNA-based vectors as well as tumor antigen-specific monoclonal antibodies (mAb). The recent clinical efficacy of these FDA-approved mAb, including cetuximab

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(anti-EGFR) and bevacizumab (anti-VEGF), has stimulated investigation into immunological mechanisms of action which may explain antitumor clinical activity.

Brief Overview of the Immune System The immune system has traditionally been divided into two major arms: innate and adaptive immunity. This dichotomy is somewhat artificial since there is tremendous interaction between the two components. Innate immunity refers to the part of the immune system that provides antigen nonspecific, first-line protection. The effectors of innate immunity include NK cells and phagocytes such as neutrophils, macrophages, dendritic cells, and monocytes that ingest extracellular debris or pathogens. Innate immunity also utilizes pattern recognition systems that recognize molecules that are not normally present in the human body: double-stranded RNA, bacterial cell wall components, lipopolysaccharide, and microbial membranes. These pattern recognition systems can take the form of enzymes like lysozyme, antimicrobial peptides (defensins), soluble factors (complement, C-reactive protein, mannose-binding lectin), and cell surface receptors (Toll-like receptors, scavenger receptors). Innate immunity is static and nonspecific, and does not change in magnitude or efficacy after repeated exposure to antigenic challenges. However, innate immune signals effectively trigger the adaptive immune system. Dendritic cells (DC) and other antigen-presenting cells link the two systems. DC ingest and process tumor antigens, after effectors of innate immunity have destroyed the tumor cell. DC then present these antigens to cytolytic and helper T lymphocytes, causing clonal expansion of antigenspecific T cells. Activation of the adaptive immune system (T lymphocytes) provides immunologic memory responses against these antigens. Thus, key effectors in tumor immunology are NK cells, B cells, T cells, and DC.

B Lymphocytes Early in the field of immunology, humoral immunity was believed to be the primary effector mechanism, in 1948 plasma cells were identified as the source of antibodies. Plasma cells are one of the two endpoints for B cells, the other being the memory B cell. B cells can be activated via T-cell-dependent or -independent antigens. Tumor antigens are T-cell dependent antigens which require binding of the antigen to the B cell receptor and a secondary activation signal via CD40 on an activated helper T cell. It is well established that B cells in cancer patients are capable of recognizing and producing antibodies to tumor antigens [1, 2]. In head and neck cancer, circulating serum antibodies have been

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found against p53 [3], MUC1 [4], p40 [5], p73 [6], and HPV E6 and E7 [7]. However, levels of circulating antibody have not been correlated with clinical outcome other than high postoperative levels of anti-p53 antibody which have been correlated with poor prognosis [8]. Interestingly, it has been noted that there is an increased frequency of IgE subtype immunoglobulins in head and neck cancer [2, 9]. The significance of this finding, if any, is unclear.

T Lymphocytes T lymphocytes were defined in the early 1960s when mice were thymectomized in an attempt to prevent lymphoma. When the initial experiments in adult mice failed to have any effect, neonatally thymectomized mice were found to have profoundly decreased lymphocyte numbers and were unable to generate antibodies despite having plasma cells. Based on these data, Miller theorized that the thymus must be the source of a “helper” cell that is required to produce antibody [10–12]. In later experiments, depletion of CD8 abolished this destruction which identified CD8 T cells as a primary effector of specific tumor/allograft rejection. T lymphocytes are defined by the presence of T-cell ­receptors (TCR) on their cell surface. TCR are part of the ­immunoglobulin superfamily and undergo germline DNA rearrangement to produce diversity much like immunoglobulin genes in B cells. TCR recognizes tumor antigens which are short peptide fragments bound to or “presented by” major histocompatibility complexes (MHC). There are two main classes of MHC: MHC I molecules found on the cell surface of all nucleated cells and MHC II is found only on professional antigen-presenting cells such as macrophages and dendritic cells. MHC class I and II binds with peptides, which are derived from tumor proteins and “processed” within the cell, and MHC then bind or present these tumor peptides on the cell surface for recognition by T cells. The TCR can only recognize peptide antigen when presented by a particular self-MHC molecule, a phenomenon known as MHC restriction, which led to the Nobel Prize in 1996 to Doherty and Zinkernagel. Therefore, CD8 T cells can recognize syngeneic (self) but not allogeneic (from someone else) tumor cells. MHC I binding tumor peptides are usually eight to ten amino acids in length, derived from endogenous proteins processed via the proteasome, and are presented to CD8 T cells. MHC II peptides are longer (11–16 amino acids), derived from exogenous proteins taken in by endocytosis, and are presented to CD4 T cells [13]. T lymphocytes are generally divided into CD4+ or CD8+ T cells. While it remains unclear how T cells are selected to become CD4 or CD8 cells, there are usually twice as many CD4 T cells as CD8 T cells released. Once antigen is ­encountered along with the appropriate costimulatory signals,

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T cells become activated and differentiated. CD4 T help (TH) cells usually differentiate into one of two major subclasses, TH1 and TH2, and this differentiation depends on the cytokine milieu in the environment at the time of activation. These two subsets of CD4 cells are differentiated by function and cytokine secretion profile. The TH1 subset is responsible for most cell-mediated immune functions such as activation of CD8 T cells, inflammation, and delayed-type hypersensitivity as well as production of complement activating IgG antibodies. Macrophages or dendritic cells will produce IL-12 in response to intracellular pathogens. IL-12 along with IFN-g(gamma) and IL-18 drive the TH1 response. TH1 cells secrete IL-2, IFN-g, and TNF-a and are felt to be the most strongly antitumor subtype. On the other hand, IL-4 drives a TH2 response [14]. The TH2 response drives B cells to produce IgM, IgE, and noncomplement-activating IgG, as well as activating eosinophils, in response to parasitic invasion. TH2 T cells are strongly implicated in allergy and are felt to be tumor permissive. TH2 cells secrete GM-CSF, IL-3, IL-4, IL-5, IL-10, and IL-13. More recently, other subsets of CD4 T cells have been identified. TH17 cells require TGF-b and IL-6 for differentiation and are defined by their production of IL-17. IL-17 is known to induce the production of several chemokines that attract proinflammatory cells and IL-17 expression is greatly increased in autoimmune diseases [15]. The final subset of CD4 T cells is the regulatory T cell (Treg) that was originally defined as a CD4+CD25+Foxp3+ T cells. Tregs are thought to be a reciprocal subtype to TH17 cells in that both are induced by TGF-b, but Tregs are immunosuppressive as opposed to TH17 cells which are proinflammatory. Tregs have recently been strongly correlated with disease status in SCCHN patients [16, 17].

Natural Killer Cells NK cells were discovered in 1975 when experiments studying tumor lysis by lymphocytes from immunized animals found lysis that was independent of previous immunization or activation [18]. This was thought to be an artifact until the NK cell was isolated and given the name “natural killer” cell for its ability to kill tumors without previous activation. NK cells kill much in the same way as cytotoxic T cells, through the interaction Fas ligand on their surface with Fas on target cells inducing apoptotic cell death. They also constitutively possess perforin and granzyme granules and degranulate causing cytolysis. Unlike T cells that are self MHC restricted and require self MHC for activation, NK cells are suppressed by the presence of self MHC via KIR receptors that inhibit NK killing when bound by self MHC [19]. These inhibitory signals can inhibit killing even when activating receptors on the NK cell are bound and therefore presentation of self MHC on the target’s surface is protective. Activation receptors

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on the NK cell include NKD2D and FcgIII receptor. NKD2D binds ligands produced by cells stressed by DNA damage or infection. FcgIII receptor is a high affinity receptor for IgG which provides a mechanism by which NK cells can recognize targets bound by antibody. Activating Fcg receptors mediate antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells, macrophages, monocytes, neutrophils, and eosinophils.

Dendritic Cells Dendritic cells (DC) are antigen-presenting cells and as such are potent initiators of the immune response. DC efficiently take up antigen via several mechanisms including phagocytosis, macropinocytosis, and adsorptive endocytosis. After uptake, antigen is shunted into lysosomes and degraded for presentation on MHC II. DC also possess B7 molecules on their surface that provide a necessary secondary activation signal to T cells after engagement of the MHC–peptide complex with the TCR. Because DC are such potent activators of T cells and initiators of adaptive immunity, they have been intensely studied as a possible therapeutic for cancer immunotherapy. Another important process mediated by DC is cross presentation of antigen derived from tumor cells or shed tumor products/vesicles. Exogenous antigen is processed via the exogenous pathway and presented to CD4 cells by DC via MHC II. However, DC are able to move exogenous antigen to the endogenous pathway and present these antigen to CD8 cells via MHC I. This surrogate presentation of exogenous antigen to the endogenous pathway is defined as cross presentation. Cross presentation serves a very important function because it allows DC to activate cytotoxic T cells against virally infected cells and tumor cells and have recently been harnessed in cancer vaccine trials.

Cancer Immunosurveillance and Immunoediting The idea of immune control of malignant cells was first proposed by Paul Ehrlich in 1908, but it was not until the 1950s that greater understanding of the immune system gave rise to a formalized hypothesis. This “cancer immunosurveillance” hypothesis was introduced by Burnet and Thomas and stated that tumor cells must have recognizably different antigens than normal cells and therefore have the potential for immune clearance. Also at that time, the phenomenon of allograft rejection via cellular immunity was observed. Because grafting of allogeneic tissue is not a naturally occurring event, Thomas proposed that the actual primary function of cellular immunity was not to protect against allografts but rather to

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protect against tumors. Conflicting experimental results led many to abandon the idea of cancer immunosurveillance for several decades, until several key discoveries have led to a revival of the hypothesis. First was the discovery of the NK cell in the late 1970s which seemed to provide innate immune protection from tumor [20]. The discovery of IFN-g and its proapoptotic effect on tumor growth gave additional support to the potential for immune clearance of cancer cells [21]. Mice lacking IFN-g receptors produced more tumors with decreased latency after methylcholanthrene challenge and addition of IFN-g was protective against transplanted, spontaneous, and induced tumors in another experiment. Studies in mice lacking perforin, a key component of cytolytic granules in T cells and NK cells, recapitulated the results in IFN-g receptor knockout mice with more frequent tumors and lower latency of formation [22]. Mice with genetically induced immunodeficiency were found to be more susceptible to both spontaneous and chemically induced tumors. In humans, epidemiologic data from AIDS patients demonstrate increased risk of lymphoma, Kaposi’s sarcoma, and virally induced carcinomas of the genitourinary tract. There also appears to be a higher risk of HPV-associated HNC in HIV+ patients [23]. These data confirm the unchallenged idea that immune protection from viral infections reduces risks of cancer associated with viruses. But what of tumors without viral etiology? Data gathered from transplant patients who are immunosuppressed to avoid organ rejection demonstrate increased risk of many tumors with no known viral etiology such as lung, head, and neck [24],

pancreatic, endocrine, colon cancer, and melanoma [25]. The cancer immunosurveillance hypothesis has given rise to the theory of cancer immunoediting which is the idea that immune surveillance of cancers provides selective pressure on tumor cells and selects for cells that can evade the immune system. One study showed that many tumors grown in immunocompromised mice are rapidly cleared when injected into immunocompetent mice, whereas cancers from immunocompetent mice continue to grow when transplanted into immunocompetent mice, indicating a qualitative difference in the cancer cells that was dependent on the immune environment [26]. The theory contends that successful tumor formation can occur only after the cancer has discovered a means by which it can evade the immune system.

Fig. 6.1  Tumor cell immune evasion and exploitation. Tumor cells secrete several small molecules and cytokines that depress NK, DC, and T-cell function and induce immunosuppressive MDSC and regulatory T  cells.

MHC downregulation and defects in the antigen presentation machinery impairs T-cell recognition. Fas ligand is expressed which kills T cells. Chemokine receptors aid in metastasis of the cancer cell to lymph nodes

Immune Escape and Immunosuppression in Head and Neck Cancer Cancer cells evade the immune system by two primary mechanisms: by reducing their innate immunogenicity or by suppressing the immune response (Fig.  6.1). Tumor cells can reduce T-cell-mediated recognition by altering HLA class I expression. It has been noted that some tumor cells have a complete loss of HLA expression due to defects in b2-microglobulin expression or function. Alternatively, chromosomal defects in the HLAencoding genes themselves can cause selective loss of HLA expression. This process has been noted in approximately 50%

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of head and neck squamous cell carcinomas [27] and was correlated with poor prognosis in esophageal squamous cell cancer [28] and laryngeal squamous cell cancer [29]. In other cancers, there is ample expression of HLA and tumor antigen but without recognition by T cells. Because HLA loss variants are killed by NK cells, one proposed explanation for the lack of NK cell killing is that cancer cells possess defects in their ­antigen presentation machinery (APM). This would reduce selectively tumor antigen-HLA peptide completely without reduction in overall surface HLA density. Endogenous antigens are processed through the ­cytoplasmic immunoproteosome which consists of various subunits including low molecular weight proteasome (LMP) 2, LMP7, and LMP10. Antigenic peptides are transported to the endoplasmic reticulum by the transporter associated with antigen processing (TAP) where they are associated with HLA class I heavy chains by tapasin [30]. Thus, SCCHN cells that express HLA I and whole tumor antigen can evade T-cell recognition through decreased expression of LMP2, TAP1, TAP2, and tapasin. The observation that T-cell ­recognition could be reconstituted with either exogenous peptide or upregulation of APM expression [31] confirms the ­biological significance of this immune escape ­mechanism. In addition to decreased expression of HLA, SCCHN tumor cells express Fas ligand which can interact with Fas and transduce a ­powerful ­apoptosis signal to activated T cells allowing immune evasion [32] by eliminating tumor-infiltrating T lymphocytes. As mentioned, decreased expression of HLA molecules is protective against T cells but increases NK cell-mediated cytolysis as the absence of HLA removes a key inhibitory signal for NK cells. Therefore, tumor cells must employ multiple mechanisms to suppress NK cell-mediated antitumor immunity. MICA, a ligand of NKG2D in NK and T cells, can be released in a soluble form to act as a competitive antagonist [33]. Cytokines and other molecules that suppress immune function such as IL-10, TGF-b, IL-6, PGE2, VEGF, and GM-CSF are known to be produced by SCCHN cells. IL-10 reduces activation of cytotoxic T cells and has been correlated with advanced stage head and neck cancer [34]. TGF- b suppresses T cell and NK activation and is a key cytokine in the differentiation of regulator T cells [35]. TGFb production is increased in preneoplastic oral cavity lesions and promotes angiogenesis and a protumorigenic microenvironment linking it to early tumor formation [36]. IL-6 signals via STAT3 to inhibit DC maturation, NK cell, T cell, neutrophil, and macrophage activation [37] and has been correlated with recurrence and survival in SCCHN [38]. Reduced DC numbers and function have been observed in this disease (Mueller-Burghaus paper). STAT3 is a transcription factor that is also involved several other immunosuppressive pathways such as IL-10 signaling [39], suppression of dendritic cells [40], downregulation of IL-12 [41], and generation of regulatory T cells [42]. PGE2 is a prosurvival, proangiogenic

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molecule that is produced by many cancers including SCCHN [43, 44]. It is also a potent immunomodulator that decreases T-cell proliferation, inhibits Th1 T cells, decreases B-cell proliferation and inhibits maturation and antigen presentation of DC [45]. VEGF, which is primarily thought of as a promoter of angiogenesis, is overexpressed in 90% of SCCHN [46] and functions to increase the ratio of immature to mature DC in the tumor microenvironment which is thought to lead to T cell anergy [47]. GM-CSF when produced in large quantities by tumors recruit myeloid-derived suppressor cells (MDSC) [48, 49] which have been identified in SCCHN.

Myeloid-Derived Suppressor Cells Myeloid-Derived Suppressor Cells (MDSC) are a diverse family of myeloid cells that are defined by Gr1+CD11b+ and in cancer patients they are usually also CD33+ and CD34+ [50]. They are increased in almost all cancer patients and, indeed, were first characterized in SCCHN [49] where their link to VEGF and GM-CSF was discovered. In addition to VEGF and GM-CSF, MDSC are induced by IL-6, IL-1b, PGE2, and complement C5a. Initial studies in SCCHN found that MDSC inhibit IL-2 secretion by activated T cells which is a key step in T-cell proliferation and escalation of cell-mediated immunity. Also, they deplete the tumor microenvironment of arginine and cysteine which are essential for T-cell activation. MDSC produce nitric oxide and reactive oxygen species that catalyze the nitration of the TCR which inhibits TCR – MHC interactions and subsequent activation. Downregulation of the TCR zeta chain which also interferes with T-cell activation is mediated by MDSC along with downregulation of l-selectin which is important for migration of naïve T cells to lymph nodes. Data on the effect of MDSC on NK cells has been conflicting with reports of both enhancing as well as suppressive action on NK cells which may be a function of the heterogeneity of MDSC populations. MDSC also promote induction of Tregs via production of IL-10, TGF- b, and arginase [50]. Treatments such as antibody depletion, retinoic acid, gemcitabine, and STAT3 blockade that diminish MDSC restore immune surveillance, increase T-cell activation, and improve efficacy of immunotherapy. The basal levels of MSDC increase with age and may contribute to increased tumor frequency and growth rate increase with age [51].

T Regulatory Cells Though it was long suspected that a subset of T cells were immunosuppressive, their characterization occurred relatively recently when it was found that this subpopulation

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were CD4+ cells that also expressed CD25 [52]. There are now four subtypes of regulatory T cells: naturally occurring thymus-derived CD4+CD25highFoxP3+ Tregs, antigen-induced IL-10-dependent Tregs (Tr1), IL-4-dependent Tregs (Th3), and antigen-specific Tregs [16]. There is also a CD8+CD25+ variant which also appears to have immunosuppressive ability but their biological significance is unclear and they are thought to be overshadowed by the much more abundant CD4+ Tregs [53]. Tregs cause anergy, apoptosis and cell cycle arrest of activated T cells via production of IL-10, TGF-b, and direct cell–cell contact [54]. They also inhibit the action of dendritic cells, NK cells, and B cells [55]. In SCCHN patients, Tregs are increased in frequency in peripheral blood and among T cells infiltrating the tumor and draining lymph nodes resulting in an immunosuppressed state [17, 56, 57]. Also, Treg numbers are inversely proportional to DC and CD8+ T-cell numbers in SCCHN [58, 59]. Treg frequency as a prognostic indicator is unclear as one study linked increased Tregs with better locoregional control [60] while another study found increased Tregs associated with early recurrence [61]. Also interesting was the finding that Treg numbers were greater in SCCHN patients after treatment than before treatment indicating that oncologic treatment increases Treg numbers [17]. These data indicate that SCCHN induces an immunosuppressed state via multiple potent mechanisms which is a ­barrier to effective cancer immunotherapy. They secrete immunosuppressive cytokines and molecules. Cytokine levels are aberrant in SCCHN patients indicating deregulation or dysregulation of cytokine pathways [62]. There is increased frequency of immunosuppressive regulatory immune cells and there is a global dysfunction of almost every facet of the immune system in SCCHN patients.

Inflammation and Cancer The strong link between inflammation and cancer is ­manifested by aberrant immune signals. The fact that some cancers arise at sites of chronic inflammation was first noted by Virchow over a century ago. Since then, chronic inflammatory states have been linked to a myriad of tumors: Helicobacter pylori infection and gastric cancer, inflammatory bowel disease and colon cancer, chronic irritation, and inflammation of the aerodigestive tract by tobacco and alcohol and SCCHN. Studies of the tumor microenvironment demonstrate infiltration of inflammatory mediators and a complex milieu of cytokines. Many of these cytokines have been previously discussed – TGF-b, IL-6, IL-10, GM-CSF – but also include cytokines such as IL-1b, IL-23, and TNFa(alpha) as well as chemokines, which are “chemotactic cytokines” that direct immune cell migration.

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Chemokines are a family of small heparin-binding cytokines that direct the movement and migration of leukocytes. There are four groups of chemokines based on the arrangement of cysteine residues near the N-terminus of the proteins: C, CC, CXC, and CX3C. The G-coupled transmembrane chemokine receptors are also divided into these four groups based on their cognate ligand [63]. SCCHN cells have aberrant expression of several chemokines. They overexpress CXCL1 which has been implicated in tumor angiogenesis, nodal metastasis, and leukocyte infiltration. CCL2 is also overexpressed in squamous cell cancer and is thought to have similar functions. CXCL5 is found in metastatic SCCHN and is involved in tumor migration and tumorigenesis. CXCL8, also found in metastatic SCCHN, promotes matrix metalloprotease secretion and subsequent extracellular matrix breakdown and tissue invasion. Of the chemokine receptors, CXCR4 and CCR7 are of particular interest as these two receptors are overexpressed in malignant cells including SCCHN cells. Increased ­expression of CXCR4 and its ligand, CXCL12, in SCCHN cells is associated with nodal metastasis, tumor recurrence, and overall survival. Studies of CXCR4 activation have shown increased metastatic potential, induction of matrix metalloprotease and collagenase expression, decreased cell adhesion and increased cell mobility. CCR7 appears to have similar biological actions. High CCR7 expression is ­clinically associated with tumor stage, lymphatic invasion, nodal metastasis and poorer prognosis [64]. A study of chemokine receptor expression differences between primary and ­metastatic SCCHN cell lines found that only CCR7 was ­consistently upregulated in metastatic SCCHN [65]. CCR7 also provides tumor survival and invasion signals via the PI3 kinase signal transduction pathway [66]. These actions in tumor cells are similar to the action of CCR7 in dendritic and CD8+ cells where they mediate chemotaxis to lymph nodes and antiapoptotic signals and may explain the ­predilection of SCCHN to metastasize to lymph nodes where there is a high concentration of chemokines. The production of chemokines and their receptors by SCCHN tumor cells represents exploitation of the immune system to promote tumor survival and metastasis. A key regulator of the inflammatory response in cancer is the transcription factor NF-kB [67] which stimulates many cancer-promoting cytokines and chemokines in SCCHN [68]. NF-kB sits downstream of several soluble factors including TNF-a, IL-1, and reactive oxygen species that are produced by macrophages and granulocytes that infiltrate tumor. Of interest in relation to SCCHN, NF-kB activation can also be elicited by cigarette smoke condensate, betel nut extract, and EGFR signaling [69–71]. Activation of the NFkB pathway induces several tumor-promoting processes in SCCHN [72]. NF-kB is traditionally thought of as a stress response transcription factor because it controls expression of several prosurvival genes such as mdm2, TRAF1, TRAF2, IAP, and Bcl-XL. These act

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as antiapoptotic signals for tumor cells and confer resistance to natural death pathways for aberrant cells. NF-kB also promotes tumor cell proliferation and expansion through regulation of a key cell cycle modulator, cyclin D1. Angiogenesis is promoted by NF-kB through VEGF production and several cytokines including TNF-a, IL-1, -6, and -8 are induced causing a positive feedback loop. Tissue invasion is promoted by the upregulation of heparinase, matrix metalloprotease, and urokinase. It has also been suggested that NF-kB mediates resistance to treatment with chemotherapy and radiation via regulation of GADD (growth arrest DNA damage) and glutathione-S-transferase [73]. The activation of NF-kB by inflammatory immune mediators demonstrates yet another subversion and exploitation of the immune system by cancer to promote key aspects of tumor formation and progression.

Cancer Stem Cells Recently, there has been growing interest in the cancer stem cell hypothesis. Heterogeneity in tumor cells has long been accepted and this theory postulates the existence of a subpopulation of tumor cells that are pluripotent and are able to effectively recapitulate the entire heterogeneous tumor when transferred to another site. They are thought known to be more resistant than other tumor cells to chemotherapy as well as radiation [74]. Several defining markers of these stem cells have been proposed. The first marker proposed was CD44 [75], a cell surface glycoprotein which binds hyaluronate but may also inhibit the action of the p53 tumor suppressor in cancer cells [76]. However, CD44 expression is abundant in normal epithelia and its utility as a cancer stem cell marker is questionable [77]. Another proposed marker is aldehyde dehydrogenase 1 which is found in many embryonic stem cells and was identified as the responsible protein in conferring resistance to chemotherapeutic agents in stem cells [78]. Because these cancer stem cells are able to reconstitute the entire tumor, many believe that ultimately, it is treatment of this small population of resistant cells that determines the success or failure of oncologic therapy. If this is the case, it is important that these cells be addressed in any treatment regimen. Because aldehyde dehydrogenase 1 (ALDH1) is not highly expressed in normal tissues, its potential as a tumor antigen target has been recently explored [79].

Immune Mediators as Cancer Biomarkers Because of the derangements in production of cytokines and other immunomodulatory molecules caused by cancer, there has been investigation into the possibility of using cytokine

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profiles as biomarkers. Biomarkers are of considerable interest because they could be useful in early detection of cancer, determination of prognosis, as a marker of treatment response and selection of optimal treatment regimen. Cytokines as biomarkers have been investigated in SCCHN in several studies. An older study found that serum TNF-a was 100fold higher in cancer patients than in disease free controls [80]. A subsequent study linking serum TNF-a levels to cancer status was published but that paper found IL-6 to be a more sensitive marker than TNF-a [81]. Another cytokine commonly cited in papers as a possible biomarker for detection of tumor is IL-8 which is elevated in recurrent or metastatic cancer [82]. In a study of over 300 subjects encompassing those with active disease, no evidence of disease and healthy smokers 60 cytokines were measured and a panel of 25 including IL-8, IFN-a, IFN-g, IL-1, and RANTES could ­correctly identify active disease with a sensitivity of 84.5% and a specificity of 92% [83]. This provided a proof-of-principle that the immune system may serve as a biosensor of malignancy and disease status. In another study, IL-6, IL-8, VEGF, and hepatocyte growth factor were elevated in cancer patients and decreases over treatment correlated with improved survival. Interestingly, elevated pretreatment VEGF was a good prognostic factor [84]. This is in contrast to a studies in nonsmall-cell lung cancer [85] and head and neck cancer (ASCO 2009 A6035) which demonstrated low pretreatment VEGF as a predictor of better treatment response and longer progression free survival. A large study of 444 patients found that high pretreatment IL-6 is an independent predictor of poor prognosis [38].

Head and Neck Cancer Immunotherapy There are several strategies for delivering tumor vaccines with each having inherent advantages and disadvantages. All methods depend on delivering an antigen to the host in an effort to elicit an adaptive cellular immune response to the tumor antigen. Most methods require the use of a specific known tumor antigen but some can use entire tumor cells as part of the vaccine to activate the immune system against multiple unspecified and unknown tumor antigens. DNA vaccines utilize delivery of naked DNA encoding a known tumor antigen to the patient. This DNA is taken up by cells and the antigen is expressed for subsequent processing and presentation by DC. DNA vaccines are safe, inexpensive, easy to deliver, and do not induce the formation of ­neutralizing antibodies allowing repeated administration. However, they have a low transfection efficiency and elicit a very weak immune response and therefore are often ­engineered to encode proteins that target DC or are given with adjuvant agents that increase DC activation. Currently in SCCHN,

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DNA vaccines encoding a HPV-16 E6/E7 fusion protein is under development for HPV positive SCCHN [86] and another vaccine encoding Hsp65 has been tested in a phase I trial [87] and demonstrated clinical response in 4 out of 14 patients with recurrent unresectable SCCHN. Bacterial/viral vaccines can deliver tumor antigen as well as functioning as an immune adjuvant because the immune system responses to a perceived infection. They are very immunogenic, relatively inexpensive, and easy to manufacture but have the downsides of potential toxicity, preexisting neutralizing antibodies, or the formation of antibodies against the bacterial or viral vector limiting repeat dosing or effectiveness. Also, these tend to elicit a stronger humoral rather than cellular immune response which is less desirable. Several such vaccine are currently under development: HPV-16 E7 Listeria vaccine [88], Vaccinia-based E6/E7 vaccine [89], and a Vaccinia-based E2 [90]. Peptide vaccines consist of synthesized peptides that have been designed to correspond to an epitope on a tumor ­antigen that binds well to the cleft of an HLA molecule. They are similar to DNA vaccines in that they are safe and inexpensive with low immunogenicity but have the added drawback of being restricted to the HLA subclass for which they were designed. The popular HLA subclass used in vaccine design is HLA-A2 as this is the most common subclass found in Caucasians. Clinical trials are underway with a MAGE-A3/ HPV-16 peptide (NCT00257738) and a LMP-2 peptide for EBV-related nasopharyngeal carcinoma (NCT00078494). To circumvent HLA restriction, whole proteins can be used as a vaccine. Whole proteins can be processed by the antigenpresenting cells and presented on self MHC to cause activation of T cells. However, the vast majority of identified tumor ­antigen proteins are self proteins and therefore the patient’s immune system is tolerant to these proteins. Therefore, there is tremendous difficulty in producing an effective immune response with protein vaccines. Tumor cell vaccines are similar to whole protein vaccines in that they are not HLA restricted and specific epitopes need not be known for their use. Often the tumor cells are given with adjuvant agents or modified by viral infection to improve their immunogenicity. A Newcastle disease virus infected tumor cell vaccine was found to induce a specific T-cell response and [91] that correlated with better clinical outcome. These vaccines tend to be labor intensive because tumor has to be isolated and processed before it can be used as a vaccine. Dendritic cells are the most potent activators of antigenspecific T cells and consequently, DC vaccines are the most widely studied cancer vaccine strategy. This is an extremely labor-intensive method in which dendritic cells are isolated from each patient and they are loaded with tumor antigen ex vivo. This loading can be in the form of peptides, proteins, DNA transfection, tumor cell lysates, apoptotic tumors, necrotic tumors, or cell fusion. After DC are loaded with

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tumor antigen, they undergo maturation and activation with various cytokine cocktails to prime them for presenting the tumor antigen to T cells. These DC are then introduced to the patients usually into the tumor or into lymph nodes. Several DC-based vaccines are currently being developed for SCCHN: intratumoral injection of DC (NCT00492947), multivalent p53 DC vaccine [92], and lysyl oxidase like-4 transfected DC [93]. There are also efforts to reverse the immunosuppression associated with cancer. One method utilizes a cocktail of multiple cytokines delivered systemically to improve immune competence. Other strategies target specific inhibitory molecules. CTLA-4 is a receptor found on T cells which sends an inhibitory signal and leads to T-cell anergy. An ­anti-CTLA-4 antibody has been developed to block this ­inhibitory signal [94]. Another inhibitory cell surface protein on T cells is programmed death-1 [95] and antagonistic antibodies to this protein have demonstrated efficacy in phase II trials [96]. Anti-KIR antibodies remove the major inhibitory signal on NK cells. There are also monoclonal antibodies which act as agonists of various stimulatory receptors such as CD40, CD137, and glucocorticoid-induced tumor necrosis factor receptor [97–99] in various stages of development.

Monoclonal Antibody-Based Immunotherapy of SCCHN Today the most widely used form of cancer immunotherapy is mAb therapy. Currently available mAbs that may have activity in head and neck cancer are listed in Table 6.1. The most extensively studied of these is cetuximab, a mouse– human chimeric IgG1 antiepidermal growth factor receptor (EGFR) mAb [100]. EGFR is an attractive target in SCCHN because it is overexpressed in 80–90% of SCCHN and leads to tumor cell proliferation, invasion, angiogenesis, tumor survival, and consequently, poor survival and prognosis [101]. The one mAb which does not target EGFR listed in Table 6.1 is bevacizumab which is a humanized IgG1 ­specific Table 6.1  Currently available mAbs for investigation or clinical use in head and neck squamous cell carcinoma Antibody Subtype Target Cetuximab Chimeric IgG1 Domain III of EGFR Panitumumab Human IgG2 Domain III of EGFR Matuzumab Humanized IgG1 Domain III of EGFR Zalutumumab Human IgG1 Domain III of EGFR IMC-11F8 Human IgG1 Domain III of EGFR Bevacizumab Humanized IgG1 VEGF-A Reprinted from Lee, S., Lopez-Albaitero, A., and Ferris, R. L. (2009). Immunotherapy of head and neck cancer using tumor antigen-specific monoclonal antibodies. Current Oncology Reports. 11 (2), 156–162. With kind permission from Springer Science+Business Media

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Fig. 6.2  Schematic representation of ADCC, the effector mAb has a constant fragment [Fc] that interacts with immune effector cells, and a variable fragment [F(ab)] that is antigen (EGFR) specific. During cross presentation, tumor antigens are degraded in the cytoplasm of dendritic cells (DC), and presented to T cells producing a cellular

immune response. Reprinted from Lee, S., Lopez-Albaitero, A., and Ferris, R. L. (2009). Immunotherapy of head and neck cancer using tumor antigen-specific monoclonal antibodies. Current Oncology Reports. 11 (2), 156–162. With kind permission from Springer Science + Business Media

against VEGF-A. A phase II trial of a combination of bevacizumab and erlotinib in SCCHN demonstrated a response rate of 14.6% and an overall mean survival of 6.8  months [46] and several other phase II trials in combination with cetuximab and pemetrexed are pending. An Eastern Cooperative Oncology Group (ECOG) phase III trial studying bevacizumab in combination with chemotherapy is also currently underway. It is becoming clear that anti-EGFR mAb mediate ­antigen-specific immune responses to targeted tumors (Fig. 6.2). There are two major mechanisms by which mAb can activate the immune system against a tumor target, direct killing via lytic immune cell (NK cell or monocytes) and complement fixation, or opsonization of tumor for phagocytosis and ­subsequent antigen processing. The latter would induce TA-specific cytotoxic T lymphocytes (CTL) to recognize and lyse tumor cells. One of the most direct methods by which antibodies can cause tumor lysis is via antibodydependent cellular cytotoxicity (ADCC) mediated by NK cells and probably monocytes and neutrophils. Panitumumab and cetuximab both mediate ADCC [102] and the extent of ADCC is heavily influenced by genetic polymorphisms in FcgRIIIa, also known as CD16 [103]. Complement activation via the classical pathway is another major effector of humoral immunity and is activated by IgM, IgG1, IgG2, and IgG3. A combination of cetuximab and matuzumab can elicit complement-dependent cytotoxicity in vitro [104]. In addition to direct activation of NK cell lysis of tumor cells, TA-specific mAbs can elicit CD8+ T-cell responses to tumor-derived antigens through interaction with FcgRs on antigen-presenting cells (APC). In human cells, there are three activating FcgRs,

FcgRI, FcgRIIa, and FcgRIII and one inhibiting FcgR, FcgRIIB [105] with FcgRIIa being the dominant receptor on APC. This antigen-specific T-cell activation was noted in 78% of patients treated with trastuzumab for breast cancer and this activation seemed to correlate positively with clinical response [106]. Specific T-cell activation has recently been demonstrated in a model using glioma and cetuximab [107] and it is likely that similar T-cell activation also occurs in SCCHN patients treated with anti-EGFR mAbs (Lee, SC and Ferris, RL unpublished data). The mechanism for TA-specific T-cell induction may actually be enhanced by ADCC and NK cell activation. In addition to their ability to mediate ADCC, activated NK cells, particularly CD56bright NK cells [108] have also been shown to secrete cytokines, such as IFN-g, TNF-a, and chemokines, such as macrophage inflammatory protein(MIP)-1a, MIP-1b, and RANTES, that inhibit tumor cell proliferation, enhance antigen presentation, and aid in the chemotaxis of T cells [103, 109]. Indeed, NK cells can ­interact with other innate immune cells that are present ­during the early phases of inflammatory responses [110]. This so-called NK cell–DC cross-talk follows the recruitment of both NK cells and DC to sites of inflammation [110, 111], resulting in potent activating bi-directional signaling. NK cells in the presence of cytokines released by DC become activated, regulating both the quality and the intensity of innate immune responses. Also, activated NK cells release cytokines that favor DC maturation and select the most suitable DC for subsequent migration to lymph nodes and efficient T-cell priming. In addition, IFN-g secreting NK cells can be recruited directly to the lymph nodes to enhance T-cell

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induction [112]. Elevated levels of the NK cell-derived chemokines IL-8, macrophage inflammatory protein-1, and RANTES have been detected within the sera of trastuzumab responding cancer patients [109]. These NK cell factors could induce the chemotaxis of naive and activated T cells, as indicated by the correlation of their presence with the infiltration of tumor tissue by CD8+ CTL. These data suggest that NK cell cytokine and chemokine production may enhance DC cross presentation and T-cell induction, with the potential to spread it to other TA [113].

Conclusion Cancer immunology is a rapidly evolving field and it is only recently that we have begun to understand the complex interaction between cancer and the host immune system. Tumor cells demonstrate several methods to exploit the immune system to help promote angiogenesis, derive prosurvival and proliferative signals, and induce metastasis and tumor progression. At the same time, cancers are able to cloak themselves from the immune system by self modification and by immunosuppression of the host. These insights and better understanding of the workings of the immune system have allowed the recent explosion of several promising immunotherapeutic agents that are currently in clinical use as well as under development.

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118 69. Anto RJ, Mukhopadhyay A, Shishodia S, Gairola CG, Aggarwal BB. Cigarette smoke condensate activates nuclear transcription factor-kappaB through phosphorylation and degradation of IkappaB(alpha): correlation with induction of cyclooxygenase-2. Carcinogenesis. 2002;23:1511–8. 70. Lin SC, Lu SY, Lee SY, Lin CY, Chen CH, Chang KW. Areca (betel) nut extract activates mitogen-activated protein kinases and NF-kappaB in oral keratinocytes. Int J Cancer. 2005;116: 526–35. 71. Bancroft CC, Chen Z, Yeh J, et al. Effects of pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal kinases on NF-kappaB and AP-1 activation and IL-8 and VEGF expression in human head and neck squamous cell carcinoma lines. Int J Cancer. 2002;99:538–48. 72. Ferris RL, Grandis JR. NF-kappaB gene signatures and p53 mutations in head and neck squamous cell carcinoma. Clin Cancer Res. 2007;13:5663–4. 73. Allen CT, Ricker JL, Chen Z, Van Waes C. Role of activated nuclear factor-kappaB in the pathogenesis and therapy of squamous cell carcinoma of the head and neck. Head Neck. 2007;29:959–71. 74. Tang C, Ang BT, Pervaiz S. Cancer stem cell: target for anti-cancer therapy. FASEB J. 2007;21:3777–85. 75. Prince ME, Sivanandan R, Kaczorowski A, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA. 2007;104:973–8. 76. Godar S, Ince TA, Bell GW, et al. Growth-inhibitory and tumorsuppressive functions of p53 depend on its repression of CD44 expression. Cell. 2008;134:62–73. 77. Mack B, Gires O. CD44s and CD44v6 expression in head and neck epithelia. PLoS One. 2008;3:e3360. 78. Chen YC, Chen YW, Hsu HS, et al. Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer. Biochem Biophys Res Commun. 2009;385:307–13. 79. Visus C, Ito D, Amoscato A, et al. Identification of human aldehyde dehydrogenase 1 family member A1 as a novel CD8+ T-celldefined tumor antigen in squamous cell carcinoma of the head and neck. Cancer Res. 2007;67:10538–45. 80. Soylu L, Ozcan C, Cetik F, et al. Serum levels of tumor necrosis factor in squamous cell carcinoma of the head and neck. Am J Otolaryngol. 1994;15:281–5. 81. Jablonska E, Piotrowski L, Grabowska Z. Serum levels of IL-1b, IL-6, TNF-a, sTNF-RI and CRP in patients with oral cavity cancer. Pathol Oncol Res. 1997;3:126–9. 82. Gokhale AS, Haddad RI, Cavacini LA, et al. Serum concentrations of interleukin-8, vascular endothelial growth factor, and epidermal growth factor receptor in patients with squamous cell cancer of the head and neck. Oral Oncol. 2005;41:70–6. 83. Linkov F, Lisovich A, Yurkovetsky Z, et  al. Early detection of head and neck cancer: development of a novel screening tool using multiplexed immunobead-based biomarker profiling. Cancer Epidemiol Biomarkers Prev. 2007;16:102–7. 84. Allen C, Duffy S, Teknos T, et al. Nuclear factor-kappaB-related serum factors as longitudinal biomarkers of response and survival in advanced oropharyngeal carcinoma. Clin Cancer Res. 2007;13:3182–90. 85. Hanrahan EO, Ryan AJ, Mann H, et al. Baseline vascular endothelial growth factor concentration as a potential predictive marker of benefit from vandetanib in non-small cell lung cancer. Clin Cancer Res. 2009;15:3600–9. 86. Yan J, Reichenbach DK, Corbitt N, et  al. Induction of antitumor immunity in vivo following delivery of a novel HPV-16 DNA vaccine encoding an E6/E7 fusion antigen. Vaccine. 2009;27:431–40. 87. Victora GD, Socorro-Silva A, Volsi EC, et al. Immune response to vaccination with DNA-hsp65 in a phase I clinical trial with head and neck cancer patients. Cancer Gene Ther. 2009;16:598–608.

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Chapter 7

HPV and EBV in Head and Neck Cancer Jeffrey Brumbaugh, Robert L. Ferris, and Shen Hu

Abstract  The focus of this book chapter is to discuss the role of human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC) and Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC). We have summarized the main events of HPV & EBV life cycle, potential mechanisms of HPV- or EBV-mediated carcinogenesis, and the implications of HPV and EBV in head and neck cancer, with an emphasis on disease diagnosis, prognosis, and therapeutic treatment. The potential of proteomics for studying these virus-associated cancers has also been discussed. A mechanistic understanding of HPV-associated HNSCC or EBV-associated NPC would require profound analysis of these tumors using advanced molecular analysis technologies, which will then facilitate the development of preventive and therapeutic strategies for these diseases. Keywords  Human papillomavirus • Head and neck squamous cell carcinoma • Epstein-Barr virus • Nasopharyngeal ­carcinoma • Proteomics

Head and Neck Cancer Cancer of the head and neck, including oral, laryngeal, and pharyngeal sites, is the sixth most common malignancy in the world. Each year, almost 650,000 patients worldwide receive the diagnosis of head and neck cancer and some 350,000 die from this disease [1]. Nearly 90% of these cancers are head and neck squamous cell carcinoma (HNSCC) in histology. HNSCC is causally associated with heavy smoking and alcohol abuse. In addition, many studies have suggested an etiological role for infection agents, such as human papillomavirus (HPV) and Epstein-Barr virus (EBV) in subsets of HNSCC, occurring mainly in the oropharynx

S. Hu (*) School of Dentistry and Jonsson Comprehensive Cancer Center, UCLA, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA e-mail: [email protected]

and nasopharynx, respectively [2, 3]. In this chapter, we will give an overview about HPV-associated HNSCC and EBVassociated nasopharyngeal carcinoma (NPC).

HPV and Its Life Cycle HPV is known as the virus that causes common warts and a host of other more serious conditions, from anogenital and aerodigestive diseases, to cervical cancer and laryngeal papillomas. On a molecular level, HPVs are circular, nonenveloped, double-stranded DNA viruses, measuring about 7.9 kb in size. They belong to the Papillomaviridae family, all of whose members have a notable similarity in genomic organization [4], and were first isolated in rabbit papillomatosis in 1933 [5]. Early studies of the virus allowed researchers to observe its life cycle, most notably the transition of the benign papillomas in rabbits as they progressed towards malignancy [6]. Today, more than 200 different types of HPVs have been isolated and there are certainly additional types that have not yet been identified [7]. The many types of HPVs are categorized into several groupings, based on tropism for infection site – cutaneous or mucosal – and on their risk for malignancy – high, intermediate, or low. The mucosal subgroup of HPVs contains more than 40 identified subtypes, making it the largest subgroup, predominantly infecting the genital and respiratory tracts [4], while the cutaneous type is mostly benign. The risk level of an HPV reflects its association with malignancy, with low-risk HPVs inducing benign hyperplasias, such as papillomas or warts, and with high-risk HPVs strongly linked to malignancy and the possibility of carcinogenesis [8]. The life cycle of an HPV virion is greatly dependent on both its own genetic mechanisms as well as those of the host cells that it infects. The genome of HPV is comprised of nine open-reading frames, which are divided into seven ­early-phase genes (E) and two late-phase genes (L) [6]. The early-phase genes encode proteins that regulate viral DNA replication, RNA transcription, and cell transformation,

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while the ­late-phase genes encode proteins that are involved in viral spread, such as the structural components of the capsid [9]. During an infection, HPVs typically target the cells in the basal layer of the squamous epithelium, integrating its genome into a host cell, and eventually replicating. First, the virus enters and infects the basal cells of the epithelium through either a wound or micro-abrasions. As these epithelial cells divide and proliferate, the viral DNA also proliferates as a low copy number plasmid, maintained in the nuclei of the daughter cells. The virus then becomes latent, exhibiting no signs of infection, for an unspecific amount of time. This latency period can last anywhere from several months to the lifetime of the host patient, and the infected tissue is both clinically and histologically normal during this time. In a subset of infected host cells, the HPV may become active, depending on the host’s stage of differentiation. Due to a strong association between the HPV and the stage of differentiation of the host cell, the HPV DNA replicates to a high copy number only when epithelial cells move from a basal position to a more suprabasal position and become terminally differentiated. It is also in these suprabasal epithelial cells that the L1 and L2 HPV proteins, which constitute the viral capsid, are synthesized and that the progeny are produced and released. Normally, the suprabasal epithelial cells would not be able to support such DNA replication, but the E1 and E2 proteins allow for productive viral DNA replication and, along with E5, papilloma formation [7, 10]. Once the dead squames of the host epithelium are sloughed off, the viral life cycle continues as the process begins anew.

Mechanisms of HPV-Mediated Carcinogenesis After an HPV virion infects a host and begins to form benign papillomas, there is a small chance that a subset of these papillomas will turn malignant. The transformation from a benign papilloma to carcinoma is a rare event, but in the case that it does occur, HPV DNA replication is ceased, and the life cycle of the virus is effectively terminated [10]. From this point, the functioning of several E genes will affect differentiation of the host epithelium, and HPV-mediated carcinogenesis can occur. While the precise molecular mechanisms of HPV-mediated carcinogenesis are not fully understood, the genome of HPV contributes a vital component to the malignancy (Fig. 7.1). Currently, high-risk HPVs are understood to contribute to carcinogenesis mainly through the actions of the two viral oncogenes E6 and E7 [11–13]. E6 and E7 are responsible for inactivating the human tumor-suppressing proteins p53 and pRb, respectively, thus allowing the potential for unchecked

J. Brumbaugh et al.

Fig.  7.1  Possible carcinogenic mechanisms of HPV in HNSCC. An active infection of HPV in the basal layer of the epithelium encodes the oncogenic proteins E6 and E7, which degrade the tumor-suppressing proteins p53 and pRb, respectively. E6 marks p53 for degradation by ubiquitination, and E7 binds and destabilizes pRb. The loss of p53 and  pRb allow for unchecked growth and eventually to malignant carcinoma

growth and a pathway towards malignancy. It has been observed that E6 proteins of high-risk HPVs bind and form a complex with p53, subsequently marking the tumor suppressant for ubiquitination and degradation [12, 13]. Conversely, small interfering RNA (siRNA) knockdown of HPV-16 E6 results in accumulation of p53 [14]. E7, on the other hand, binds and destabilizes the Rb tumor suppressor protein and related proteins [11, 15]. Accordingly, it has been suggested that E6 is mainly responsible for offsetting the increased levels of p53 and E7 produces a necessary function in promoting cell-cycle progression and viral DNA replication in differentiated keratinocytes. It has also been theorized that E6 may not assist in complete p53 degradation, but merely diminish its effects [16]. Though the exact role of E4 has yet to be determined, its RNA has been detected most abundantly in benign HPVinduced papillomas, implying that it may play a significant role in the life cycle of the virus and perhaps in HPV-mediated

7  HPV and EBV in Head and Neck Cancer

carcinogenesis [10]. The E4 protein is found exclusively in the differentiating layer of the host epithelial cells and promotes the collapse of the cytokeratin network. Similarly, the precise role of E5 in carcinogenesis is not completely clear, but it may have an important part in stimulating the transformation of HPV and promoting the proliferation of HPV-infected cells [4]. Low-risk HPVs have not been studied as thoroughly as high-risk types, due to their infrequent role in HPVmediated carcinogenesis. The E6 and E7 oncogenes of low-risk HPV types also target p53 and pRb, but with less ability to perturb their host’s cellular functions than highrisk types [16]. Consequently, they have less capability of inducing carcinogenesis. By any means, the molecular mechanisms of HPV-mediated carcinogenesis are far from being completely understood, and additional studies are warranted. To compound this lack of knowledge, it may also prove difficult to separate the molecular mechanisms and etiological role of HPV in carcinogenesis from the many cofactors of the disease. Studies have suggested HPV is not a sufficient cause for cancer, only recognized as a necessary one [4, 17]. For instance, evidence suggests that the transformation of HPVinfected cells into malignancies require cellular mutations as an impetus, such as carcinogenic agents like tobacco or UV irradiation [10]. Several studies have even shown that HPV-transformed cells are non-invasive, implying the need for a cellular cofactor to be present in order for carcinogenesis to occur [4].

HPV in Head and Neck Precancer Commonly, HPV itself is recognized in the context of carcinogenesis as the virus is associated with cervical cancers as well as several other anogenital carcinomas. However, recent clinical studies have presented convincing evidence for a causal role of the virus in a subset of HNSCCs [3, 18]. The HPVs of most concern in HNSCC are the mucosal, high-risk types that can infect the epithelium of the aerodigestive tract. Most frequently, HPV-16 and, to a lesser extent, HPV-18 have been detected and identified as two such types, playing important roles in head and neck carcinogenesis [19]. Since HPV infection of the cervix follows a genetic progression from benign papillomas to malignant lesions, the detection of HPV in the precancerous lesions of HNSCC may be an important indicator of the potential presence of the disease. In the precancerous stages of HNSCC, dysplastic lesions undergo a series of molecular and genetic alterations that eventually lead to malignancy. In order to implicate HPV with an etiological role in head and neck carcinogenesis, it is important to know whether the prevalence of HPV DNA

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present in early dysplastic lesions increases as malignancy develops. For this purpose, numerous studies have measured the HPV DNA in premalignant lesions and the reported results are conflicting, with HPV prevalence ranging from 0% to 88% [20–23]. The observed discrepancies may be attributed to the variation in examined samples and the sensitivity of the applied methodologies. Overall, HPV may in fact play a role in precancerous lesions, but to date this has not been histologically or morphologically defined.

HPV in Head and Neck Cancer Though the evidence of HPV involvement in precancerous lesions is varied, the evidence of HPV in HNSCC is now well-established. HPV-positive HNSCCs comprise a heterogeneous group of squamous cell carcinomas (SCCs) of the oral cavity, pharynx, and larynx [24], each with varying ­biological/clinical characteristics and unique etiology. Specifically, oropharyngeal and tonsillar carcinomas have emerged as an area of particular interest due to their notably strong association to HPV. A systematic review of the data from 60 published studies revealed an overall HPV prevalence of 25.9% in HNSCC based on a total of 5,046 cancer specimens examined. HPV prevalence was found significantly higher in oropharyngeal SCCs (35.6% of 969) than oral SCCs (23.5% of 2,642) or laryngeal SCCs (24.0% of 1,435). HPV16 accounted for a larger majority of HPV-positive oropharyngeal SCCs (86.7%) compared with HPV-positive oral SCCs (68.2%) and laryngeal SCCs (69.2%). HPV-18, on the other hand, was very rare in HPV-positive oropharyngeal SCC (2.8%) compared to other head and neck sites of oral SCCs (34.1%) and of laryngeal SCCs (17.0%) [25]. A recent case-control study of 100 patients with newly diagnosed oropharyngeal cancer and 200 control patients without cancer concluded that HPV-16 DNA was detected in 72% of 100 paraffin-embedded tumor specimens, and 64% of patients with cancer were seropositive for the HPV-16 oncoprotein E6, E7, or both [26]. To further separate HPV-positive oropharyngeal SCCs from other HNSCCs, known risk factors for the disease seem to be markedly absent [27]. Thus, increasing evidence suggests that HPV-associated oropharyngeal carcinomas are in fact a separate malignancy, distinct from other HNSCCs in terms of both risk factors and biology [27, 28]. The distribution of specific HPV type and infection sites in laryngeal papillomas and tonsillar infection in oropharyngeal SCC may suggest “specific virus-tissue interactions” that only allow for HPV infection in certain sites of the head and neck [25]. While HPV-16 and HPV-18 play a significant role in oropharyngeal carcinogenesis, HPV-6 and HPV-11 may play an analogous role in laryngeal papillomas [19].

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Although more than one type of HPV can be found in tumor specimens, the low-risk types of HPV found in the majority of laryngeal papillomas differ from the high-risk HPVs in oropharyngeal SCCs. Similarly, the split between the types of HPV found in oropharyngeal and laryngeal SCCs is evidenced by the uneven distribution of HPV-16 and HPV-18 as previously mentioned. In addition, data collected from oropharyngeal SCC studies have shown that the tonsils are infected in particular more often than the rest of the oropharynx, though both contain HPV-16 as the dominant virus [25]. It seems that laryngeal papillomas and tonsillar SCC point towards a specific HPV-tissue interaction, but further investigations are required to determine a more precise distribution of HPV types in various HNSCC locations. High risk HPV subtypes may also possess more potent immune evasion capability, permitting malignant progression. Today, more than 20 different types of HPVs have been reported in HNSCC, and as many as 14 have been identified as high-risk in cervical cancer [25]. In 2005, the International Agency for Research on Cancer (IARC) concluded that there was sufficient evidence to implicate HPV-16 in causing carcinomas of the oral cavity and of the oropharynx, limited evidence for HPV-18 in the oral cavity, inadequate evidence for other HPV types in the oral cavity and in the oropharynx, limited evidence for HPV-6, -11, -16, and -18 in the larynx, and inadequate evidence for the carcinogenicity of HPV in the esophagus [24].

Detection of HPV and Diagnosis of HPV-Positive HNSCC In the study of HPV in human cancers, many detection techniques of HPV DNA have been established. One of the primary concerns when performing molecular detection of HPV DNA is the sensitivity and reliability of the applied techniques. Compared to other HPV-positive SCCs, such as cervical carcinomas, HPV-positive HNSCCs and related dysplasia seem to have a relatively low level of HPV DNA present. For instance, studies involving polymerase chain reaction (PCR) assays of HPV-positive HNSCCs have produced weaker results when using samples from both oral mucosa and cancerous lesions in the head and neck as opposed to cervical carcinomas, potentially due to saliva clearance of the virus [28]. A wide variety of methods are currently being used to detect HPV in HNSCC, including PCR assays, in situ hybridization, Southern blot, and antibody detection, which in turn may provide for early diagnoses and treatment of HNSCC. PCR is utilized as one of the most sensitive methods of detecting HPV DNA in both cancerous and precancerous lesions. However, one of the drawbacks of using PCR is also

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one of its most pronounced strengths: its extreme sensitivity. As such, PCR is prone to contamination, and if contaminated, a sample of cancerous tissue analyzed using PCR may provide either an overestimation or underestimation of possible HPV positivity. Commonly, PCR detection of HPV relies on the amplification of the E6, E7, and L1 sequences of the viral genome. Compared to conventional PCR techniques, real-time quantitative PCR analysis is able to quantify the amount of HPV DNA in a tissue sample as well as greatly reduce the risk of contamination, thus providing more accurate results [17, 22, 29]. Recently, a “MassARRAY” assay based on coupling mass spectrometry with competitive PCR, was described for measuring HPV DNA in serum and/ or peripheral blood fraction of individuals with cervical, head/neck, or bladder cancers. The technique may be more sensitive than real-time quantitative PCR-based assays, while specificity was maintained [30]. Before PCR was widely used, in situ hybridization (ISH) and Southern blot were prevalently used for detecting HPV DNA. In situ hybridization (ISH) involves the use of typespecific radioactively labeled DNA probes complementary to HPV sequences for detection. It is a clinically useful test to confirm the diagnosis of HPV and therefore has widespread applicability [8]. Southern blot, on the other hand, is known for its high specificity and low rate of contamination, even being able to distinguish between integrated and episomal HPV DNA [6]. Aside from the more classical methods of HPV detection, screening individuals for the presence of HPV antibodies or related proteins provides yet another way to detect the virus. HPV infections are in fact very commonplace among adults, and as a result, anticapsid antibodies are produced during the infection. Those exposed to harmless HPV infections and those exposed to high-risk carcinogenic HPVs, however, differ in the type of antibodies that the body produces. Serum antibodies to the viral capsid proteins, E6 and E7, are most frequently detected in individuals with HPV-positive cancer, as opposed to the anticapsid (L1) protein antibodies found in normal individuals. The detection of such specific immunologic biomarkers may provide evidence for the presence of the viral genome in patients as well as indicating a higher risk of developing HPV-mediated carcinoma. Similarly, immunohistochemical analyses of the expression of p16 may provide an important method of detecting productive HPV infection [27, 31, 32]. In many types of SCCs, the functional loss of p16 has been observed. In contrast, HPV-positive SCCs, including those of the head and neck, have shown a strong overexpression of p16, probably due to the impairment of the negative feedback control of pRb by the viral oncogene E7 among other mechanisms [27, 31]. Further investigation of p16 may support its potential application as a biomarker in standard screenings, reflecting HPV status in early dysplastic lesions [27].

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Early diagnosis of HNSCC is critical for reducing the rate of mortality of the disease and is the focus of much ongoing research in the field. As such, the molecular detection of HPV serves a vital purpose. Often times, it may be difficult to appropriately diagnose HPV-positive HNSCC, since there are such a wide variety of molecular assays, sampling methods, and oral specimens available that standardization of methods is a difficult task [28]. Nevertheless, a positive detection of HPV infection does not necessarily indicate the development of head and neck cancer. In this regard, it is also important to develop new molecular biomarkers (in addition to HPV DNA and proteins) for an improved diagnosis of HPV-positive HNSCC.

Prognosis of HPV-Positive HNSCC and Therapeutic Treatment Prospective clinical trials and large retrospective studies have shown that patients diagnosed with HPV-positive HNSCC have a more favorable prognosis than patients who have HPV-negative HNSCC [31, 33–36]. It has been estimated that HPV-positive tumors may reduce the risk of death by nearly 60–80% in HNSCC patients when compared to HPVnegative tumors [8]. Since HPV-positive HNSCCs are molecularly and clinically distinct from HPV-negative cases, many hypothesize that there are factors specific to HPVpositive tumors that can explain the reduced rate of mortality and that cause them to respond differently to treatment [31]. This certainly warrants further molecular analysis of HPVpositive HNSCC to understand the molecular mechanism responsible for favorable prognosis. Within the past few years, vaccines have come to the forefront of the battle against virus-associated cancers. A vaccine that could potentially prevent HPV infection, suppress its viral effects, or both, would prove effective in treating HPVpositive HNSCC. Theoretically, a prophylactic vaccine should prevent HPV from infecting a host epithelium by completely neutralizing the virus upon exposure. Several prophylactic vaccines are already on the market (e.g., Gardasil and Cervarix) [7]. Such vaccines have the potential to prevent a significant number of anogenital carcinomas, most notably cervical cancer, but their effectiveness in preventing HNSCCs still remains to be evaluated [18]. In addition, the duration of protection that the vaccine offers is unknown, and they do not guard against all types of HPV that could potentially result in carcinogenesis [7]. If a patient has already been infected with the virus, a therapeutic vaccine should instead induce a cellular immunity in which mainly T-cells are primed against HPV antigen epitopes expressed by oncogenes E6 and E7 [9]. It may also be possible to develop a vaccine that provides both types

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of protection from HPV: prophylactic and therapeutic. Chemotherapeutic vaccines targeting the viral oncogenes E6 and E7 are still under development today. Similar to the idea of a therapeutic vaccine targeting E6 and E7, gene therapy for HPV-positive carcinomas provides another possible tool in combating HPV-mediated carcinogenesis. While certainly far from clinical use in humans at the moment, the potential implementation of gene therapy might entail the use of E6 short interfering RNA, antisense RNA to E6 and E7, and a mutated E2 protein that would induce apoptosis in cancer cells [9, 10, 28]. Other studies have even suggested that altering the metabolism of estrogen in the body could prevent some laryngeal papillomas and laryngeal cancers, since estrogen levels can affect the risk of cancer in some tissues sensitive to hormones [10]. Radiation therapy has proven to be rather effective in treating HPV-positive HNSCCs with significantly improved survival rates of HPV-positive carcinomas in the head and neck [37, 38]. Cidofovir is an antiviral drug used to treat HPV-induced laryngeal papillomatosis and other viral infections, with initial reports suggesting activity in cervical carcinoma cells. In the presence of Cidofovir, HPV-16-transformed HNSCC cells exhibit a pronounced sensitivity to irradiation, perhaps due to the induction of p53 expression by Cidofovir. Because p53 mediates pro-apoptotic effects of XRT, this provides a mechanistic explanation for Cidofovir as a radiation sensitizing agent [39]. Immunotherapy (e.g., targeting p53-derived or E7-derived peptides) may provide a potential approach to combating HPV-associated HNSCC [40, 41]. Wild-type sequence (wt) p53 peptides are attractive candidates because elevated levels of p53 protein occur in a high proportion of human carcinomas, including HNSCC. However, in HPV-associated HNSCC, increased proteasomal degradation of p53 may result in appreciable presentation of p53-derived peptides, despite low p53 expression. The requirement of p53 overexpression would visually exclude these individuals from wt p53-based immunotherapy. In fact, both wt and mutant p53 molecules were found sensitive to E6-mediated degradation in HPV-associated HNSCC and that this HPV-induced p53 degradation was correlated with increased T-cell recognition of the tumor cells in vitro and in vivo. These findings suggest that p53 peptides may be useful tumor antigens for HNSCC immunotherapy and T cell-mediated immunotherapy against wt p53 should not be restricted to tumors overexpressing p53 [41]. HPV-encoded oncogenic proteins, such as E7, are also promising tumor-specific antigens. T-cell frequencies against E7 derived peptides (HPV-16 E711–20 and E786–93) were found significantly elevated in HPV-16 positive HNSCC patients compared with HPV16negative patients or healthy volunteers. In addition to the presence of HPV-specific effector T cells, successful tumor elimination requires that HPV-infected tumor cells function

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as appropriate targets for CTL (cytotoxic T lymphocyte) recognition and elimination. The study also suggested endogenous E7-specific immunity exists even in the presence of ongoing virus-associated malignancy, perhaps due to immune escape of tumor cells from CTL recognition by downregulation of some antigen-processing machinery component expression. These findings support that E7-derived peptides are potentially useful targets to facilitate HPV-specific immunotherapy of HNSCC [40].

EBV and Its Life Cycle Nearly 45 years ago, EBV was first discovered in 1964 from a patient with African Burkitt lymphoma (BL) [42]. As one of the most common human viruses, EBV is known today around the globe, infecting adults and children alike. Spread from person to person through close contact, EBV infection usually goes unnoticed by most, occurring as a subclinical illness or simple childhood sickness. The age at which a person becomes infected with the virus, however, depends on several factors, including living conditions, hygiene, and sexual behavior. By adulthood, over 90% of the population has been infected by EBV at some point in their lives [43]. EBV itself is a g(gamma)-herpes virus and a member of the Herpesviridae family. The herpesviruses consist of generally large, complex DNA viruses, able to encode about 100 different proteins, and are one of the largest virus groups that significantly infects the pediatric population. After a primary infection, herpesviruses typically establish permanence in their host, in the form of a life-long infection. In the case of EBV, the virus perpetuates its existence by latently infecting circulating B-cells, which are subsequently shed into genital and salivary secretions. Instead of damaging or destroying the B-cells that it infects, EBV increases the number of B-cells in the host and extends their survival, causing a sudden growth of infected cells and ensuring the virus’ permanence [43, 44]. The life cycle of EBV consists of two separates phases, which include an active, lytic form of infection and a latent state of infection. Most often, EBV resides in its host in a state of dormancy, infecting B lymphocytes in the blood. In this state, EBV expresses very few viral proteins and remains undetectable by the host immune system. Each B-cell carrier would contain around 2–5  copies of intact, circular viral DNA. But, as a highly infectious virus, EBV is capable of periodically reactivating and commencing the lytic phase of its life cycle. The lytic cycle then produces new progeny virions, infects more B-cells, and eventually returns to a state of latency. Since the life cycle of EBV so closely resembles the natural differentiation pathway of antigen-activated B-cells, the virus is able to guide infected B-cells through its various

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stages of differentiation, essentially dictating whether EBV will exist in its latent or active form. It is in its ability to alter the various stages of B-cell differentiation, to permanently affect its growth transformation, that EBV has its pathogenic capacity, which in turn results in the numerous lymphomas and carcinomas for which EBV is responsible [44–46].

Mechanisms of EBV-Mediated Carcinogenesis While it may seem counterintuitive, EBV poses the larger risk of becoming tumorigenic when in its latent state, rather than in its active state. When EBV induces growth transformation in its host cell, the production of progeny virions is ceased, and the virus undertakes a tumorigenic pathway of replication. The host B-cells propagate EBV’s DNA by replicating it as an extrachromosomal episome, utilizing the host’s own DNA polymerase. The tumorigenic properties of this type of latent infection largely come from a small set of latent genes, which include the latent membrane proteins (LMP1, LMP2A, and LMP2B) [47, 48], EBV nuclear antigens (EBNA1, EBNA2, and EBNA3) [49, 50], and the EBV encoded noncoding RNAs (EBERs) [46, 51, 52] (Fig. 7.2). EBNAs play a major role in promoting the activities of the other proteins, primarily oncogenic LMPs. In particular, EBNA1 holds a great deal of significance since it is found universally in all EBV-associated tumors and the presence of EBNA1 enables the EBV genome to be replicated and passed along to the daughter cells of an activated, dividing host. In addition to EBNA1, EBNA2 is produced during an infection and acts as the major transcriptional regulator of both cellular and viral expression. It has been shown, by deletion of the gene encoding EBNA2, that the protein is crucial in the transformation of infected B-cells [46, 53, 54]. Functionallyspeaking, EBNA2 upregulates the expression of several B-cell antigens, such as CD21 and CD23, plus the viral membrane proteins LMP1 and LMP2. Lastly, the EBNA3 family – which includes EBNA3A, EBNA3B, and EBNA3C – encode hydrophilic nuclear proteins. The EBNA3s, with the exception of EBNA3B, have been demonstrated to be indispensable in B-cell transformation in vitro [43, 46, 53, 54]. As the principal EBV oncogene, LMP1 is necessary for cell immortalization and has demonstrated transforming ability. This viral protein has a significant effect on epithelial cell growth and inhibits cell differentiation, often inducing growth transformation. LMP1-positive cells have increased mobility, which in turn leads to greater tumorigenic potential and faster disease progression. In addition, LMP1 is also involved in suppressing immunogenic responses through its capacity to downregulate T-cell response genes related to tumor antigen presentation. On the other hand, less is known

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play a more critical part in contributing to pathogenesis, especially in initiating B-cell growth transformation [46, 53]. The EBNAs, LMPs, and EBERs have been identified as the molecules of most interest in EBV-associated tumorigenesis. However, to some extent, EBV infection appears to be necessary but not sufficient for tumorigenesis in NPC. Although there are many aberrations that contribute to tumorigenesis, the critical signals in NPC development are the Wnt pathway, transcription factors NF-kappa B and beta-catenin. Most NPC tumors exhibit Wnt pathway protein dysregulation and over-expression of beta-catenin and NF-kappa B [43]. As with any carcinoma, the loss of tumor suppressors is to be expected. In EBV-mediated tumorigenesis, however, levels of tumor suppressors are less predictable. While p16 and p27 activity is decreased in EBV-associated carcinoma, high levels of p53 are found. It is unclear whether increased p53 levels contribute to EBV malignancy or whether it is merely a natural response to infection. Another oncogene that may play a role in EBVassociated carcinomas is the BARF1 gene, which has been demonstrated to play an important role in growth promotion. Since the BARF1 protein exists in serum, it may also prove to be a useful diagnostic biomarker in some patients with EBV-associated carcinoma [43, 55, 56].

EBV in Nasopharyngeal Precancer Fig. 7.2  Possible carcinogenic mechanisms of EBV in NPC. After the primary infection of a B-cell by EBV, the growth-transformed B-cell may undergo two different pathways. Usually, EBV will establish a latent infection in the B-cells, lying in a state of dormancy. On occasion, however, the lytic phase of its lifecycle may commence, and EBV will replicate in the B-cells, shedding EBV virions which can then latently infect either more B-cells or the epithelium of the nasopharynx. As the EBERs maintain viral latency, the EBNAs upregulate the LMPs – namely LMP1, the principal oncoprotein responsible for inhibiting cell differentiation and promoting malignancy

about LMP2A and LMP2B. Studies in rodent populations have suggested that LMP2A is a driving force behind the proliferation and survival of B-cells, thus maintaining EBV latency and preventing the activation of the EBV lytic cycle. Recent reports have also shown that LMP2A can transform epithelial cells. The role of LMP2B is less complex, and it is thought to regulate LMP2A function [43, 46, 54]. Lastly, the presence of EBERs is a characteristic of latent EBV infection, although they are not necessary for B-cell transformation. The EBERs are small, nuclear RNAs that are  the most abundant RNAs in EBV-infected cells. They are present in all forms of latency and are thought to contribute to malignancy by maintaining viral latency. In some EBV-associated malignancies, such as BL, EBERs seem to

NPC is an EBV-related malignancy found mainly in parts of Southeast Asia. Tumorigenic activities of EBV in NPC have been well studied and documented, but there is a glaring lack of evidence regarding the interaction of EBV and precancerous lesions of NPC. In contrast to many cancers, early manifestations of malignancy such as dysplasia, or carcinoma in situ are rare in the development of NPC. In one study, screening for dysplasia and carcinoma in situ, only 11 out of over 5,000 nasopharyngeal-biopsy samples displayed early malignant changes without adjacent invasive carcinoma. These 11 samples were then analyzed for the presence of EBV, and in all cases, the expression of EBV-DNA, EBERs, and LMP1 were detectable. These results seem to imply that the preinvasive lesions serve as a focal point of EBV-induced cellular proliferation, and that EBV infection precedes the development of malignancy [57]. However, in a similar study, EBVDNA was detected in only a portion of the cells of tissue samples with carcinoma in situ, as opposed to all of the cells [58]. This may suggest that EBV infection occurred after the initial neoplastic event, and that some preceding genetic change may affect viral infection, allowing for a latent EBV infection to establish and express oncogenic proteins. Another study highlights the loss of the p16 tumor suppressor as a potential contributor to the progression towards ­invasive

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malignancy. A more recent study suggested that chromosomal losses, which affect the chromosome 3p occur at a preinvasive stage, early in the development of tumorigenesis. Early dysplastic lesions examined in this study supported the evidence that EBV infection in fact occurs after genetic alterations in the cell, allowing a latent EBV infection to develop [59]. Of course, these controversial results warrant further studies to investigate the role of EBV in precancerous lesions of the nasopharynx. Other cofactors in addition to EBV infection, including genetic modifications in tumor suppressors such as p53 and pRb or in ras genes, as well as environmental factors, may need to be considered as possible sources of preinvasive malignancy.

EBV in Nasopharyngeal Cancer The association of EBV with NPC can be dated back to the early 1970s [60–62]. While NPC is often simply thought of as an EBV-related malignancy, it can be defined more precisely as a squamous cell carcinoma (SCC) that develops around the ostium of the Eustachian tube in the lateral wall of the nasopharynx. NPC tumors are comprised of malignant, EBV-infected epithelial cells that are surrounded by reactive lymphocytes [63]. The  World Health Organization (WHO) classifies NPC into three categories, based on its histology. Type 1 – keratinizing SCC – is characterized by well-differentiated cells that produce keratin. Type 2 – nonkeratinizing SCC – is more varied in  cell  differentiation and does not produce keratin. Lastly, type 3 – also nonkeratinizing SCC – is undifferentiated with highly variable cell types. In NPC, types 2 and 3 are EBVassociated and have an overall better prognosis than type 1, which is typically EBV absent [43, 63]. Some studies suggest that regardless of subtype, all NPC show strong evidence of EBV as an etiological factor in the onset of the disease, whereas others maintain that the association of EBV with more than type 3 NPC is controversial at best [43, 64, 65]. EBV episomes and viral proteins are consistently detected in all cells of most tumors associated with the virus, ­implying the necessary nature of EBV in the development of these malignancies. Since a ubiquitous EBV virion can lead to such a wide ranges of cancers, it is clear that other factors aside from EBV must influence the development of these cancers as well. As such, EBV is recognized as a necessary, but insufficient, cause for NPC. EBV strain variation may play a part in determining the type of cancer that will arise, implying specificity in EBV strain and malignancy. Potential cofactors, including epidemiological patterns, genetic susceptibility, and environmental factors such as salted or ­pickled foods and exposure to fumes and chemicals from the occupational environment have also been associated with

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the development of NPC [62, 64, 65]. In areas with high i­ncidence, NPC clusters in families which suggests that both geography and genetics may influence disease risk. A genome-wide scan for familial NPC revealed evidence of a major susceptibility locus for NPC on chromosome 4 [66]. In type 3 NPC, EBV infects the epithelial cells of the posterior nasopharynx. To explain the infection of these specific cells by NPC, two mechanisms have been proposed. First, while an EBV-compatible receptor on epithelial cells has not been found, the CD21 receptor, which is a surface protein antigenically related to B-cells, could potentially be used as a point of virion entry. Alternatively, it has been suggested that EBV may gain entry into the nasopharyngeal epithelial cells through IgA-mediated endocytosis [67, 68]. In either case, the EBV genomes present in the epithelial cells are of clonal origin, and EBV is distinctly absent from surrounding tissues and invading T-lymphocytes. Another point to consider when discussing the causality of EBV in NPC is the way in which EBV-infected cells can evade the immune response. EBV-infected epithelial cells in the nasopharynx possess normal antigen processing and are recognized by EBV-specific cytotoxic T-lymphocytes, but they are not destroyed [44, 64, 69]. One possible explanation involves the increased production of IL-1a(alpha) and IL-1b(beta) by the infected epithelial cells, which control the levels of lymphocytes and contribute to the growth of the tumor [70]. In addition, the over-expression of bcl-2 allows the infected cells to bypass apoptosis and this contributes to oncogenesis [71]. Overall, it has been well-established that EBV contributes to the development of NPC, although to which specific WHO classification is less clear. EBV has been consistently linked to the disease in epidemiological studies, serological analyses, and the expression of malignant viral products by EBV, implying an etiological role for the virus in NPC.

Detection of EBV and Diagnosis of EBV-Positive NPC The detection methods for EBV mainly rely on the presence of EBV-DNA and its gene products [72, 73]. Depending on the type of latent infection present, different EBV-associated proteins may be detected in EBV-infected patients. Typically in NPC, type 1 and type 2 latency are observed [46, 74]. Namely EBER transcripts, as well as ENBA1 and LMP2A proteins, characterize type 1 latency, while type 2 latency additionally expresses LMP1 and LMP2B [73]. Lab testing of EBV can be accomplished in several ways, including in situ hybridization, Southern blot analysis, EBV-DNA amplification with PCR, and serological analysis, all of which may contribute to early detection and diagnosis of EBV-positive NPC [73].

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EBER in situ hybridization is considered the gold standard for detecting and localizing latent EBV in tissue samples. EBER transcripts are expressed in virtually all EBV-related NPC tumor cells yet are notably absent in adjacent normal tissue. This localization appears to occur in the early stages of infection, and as such, becomes a valuable diagnostic tool. The main advantage of using in situ hybridization is its ability to localize EBV in the context of cytological and histopathological features of the tissue [56, 73]. Southern blot analysis is based on the variable number of terminal repeats at the ends of each EBV-DNA molecule. Since any cell is only infected once with EBV, each infected cell may contain up to 20 terminal repeats from the infecting genome of the virus. EBV-related NPCs harbor monoclonal EBV-DNA that can be detected with a clonality assay. After lesional EBV-DNA is subjected to BAMHI restriction enzyme, electrophoresis, and transfer, monoclonal patterns can be distinguished and the amount of linear EBV-DNA present can give some indication of active viral replication [73, 75]. EBV-DNA amplification with PCR provides yet another method for the detection of EBV-DNA in blood, fluid, or ­tissue samples [73, 76]. Since EBV-DNA is present to some degree even in healthy virus carriers, this detection method lacks the specificity of EBER in situ hybridization. The use of EBV-DNA amplification with real-time quantitative PCR, however, leads to the possibility of EBV viral load measurement. The assays are relatively quick, can be used as a screening method based on body fluid testing, and therefore appear to have advantages over other methods of viral detection [73, 77]. Since quantitative PCR permits precise measurement of EBV-DNA levels in clinical samples, EBV viral load assays might be able to distinguish low-level infection in carriers from higher levels associated with EBV disease. Serology remains the most accurate detection method for confirming acute versus remote EBV-related infections. EBV-specific serological assays through enzymelinked immunosorbent assay or immunofluorescent assay are used for more precise indication of acute or recurring EBV infection [73, 78]. Since other diseases can present biomarkers similar to those associated with EBV-related NPC, additional detection methods are commonly used to confirm the presence of EBV, most notably quantitative DNA amplification assays. The early detection of EBV in NPC is absolutely critical concerning the prognosis of a patient, since NPC exhibits an extraordinarily high cure rate for early stage disease. The detection of NPC is based on the clinical history of the patient and a physical examination, but a definitive diagnosis requires a biopsy of the lesion. A combination of radiologic assessments, including CT and MRI scans of the head and neck, are currently used to assess the tumor and stage of the

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disease. Although there are mixed reviews of the usefulness of serology in predicting and diagnosing NPC, it is often a ­common technique to determine the status of EBV infection and site of the primary tumor [79].

Prognosis of EBV-Positive NPC and Therapeutic Treatment Early diagnosis of NPC is vital in combating the disease, as it is much more effective when the tumors are treated at early stage. With traditional radiotherapy or chemotherapy, early stage NPC treatment has proven to be highly effective, while later stage treatment using the same therapies, targeting NPC that is already metastatic or recurrent, provides much less favorable results [80]. The prognosis for individuals with NPC recurrence or progression remain very dim, as about 85% of patients die within 1 year, and virtually all die within 3 years [81]. Considering the poor prognosis of individuals diagnosed with late-stage NPC, it is important to screen patients regularly for the presence of the disease in order to provide effective treatment. Similar to other carcinomas, the prognosis of NPC depends on the size of the tumor, lymph node involvement, and distant metastasis [43]. Several studies have attempted to characterize the prognoses of patients with EBV-positive NPC in relation to the presence of several different diagnostic biomarkers [82, 83]. One study has demonstrated that the presence of EBNA1 DNA in peripheral-blood cells is an important risk factor for patients with NPC, indicating a significantly higher risk of developing distant metastasis and an overall lowered survival rate [83]. Another study has suggested that the quantitative analysis of plasma EBVDNA levels is a useful tool in screening and monitoring potential NPC patients [84]. The standard treatment for NPC is radiotherapy, but better prognoses are obtained when utilized in combination with adjuvant chemotherapy [43]. Since EBV infection in tumor cells is generally restricted to a latent form, switching from the latent form of viral infection into the lytic form may induce tumor cell apoptosis [85]. One potential tool for accomplishing this is the use of valproic acid (VPA), an antiseizure drug that also has strong histone deacetylase inhibitory activity, for activating lytic viral gene expression in EBV-positive tumors [86]. Another line of study involves the use of a variety of chemotherapeutic agents, including cisplatinum, 5-fluorouracil (5-FU), and taxol to induce the switch from the latent to lytic form of EBV infection in tumor cells. Because the lytic form of EBV infection converts the cytotoxic prodrug, ganciclovir (GCV), into its active form, the combination of GCV and chemotherapy has

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been shown to be much more effective in the treatment of EBV-positive NPC than either agent alone [87]. A follow-up study of the metastatic NPC patients with chemotherapy indicates that a high percentage of the patients (~70%) can attain complete responses and long-term survival (diseasefree for at least 36 months). The data confirms the promising potential of chemotherapy in treating NPC [82]. Cell therapy is another therapy that holds great promise for a specific treatment against EBV-positive NPC, targeting the viral aspect of the disease. EBV is present in virtually all poorly differentiated and undifferentiated nonkeratinizing NPCs, which makes it a reliable target in cell therapy. EBV expresses a restricted set of viral antigens, namely LMP1 and LMP2, in addition to EBNA1, all of which are immunogens that are capable of inducing a T-lymphocyte response. Because it has been shown that NPC cells are capable of immunologic processing for cytotoxic T-lymphocyte recognition, studies have been conducted to explore the possibilities of pulsing dendritic cells with EBV-peptides to enhance T-lymphocyte immunity. Generally, clinical responses to cell therapy have been well tolerated, although it does have its limitations in tumor specificity and targeting tumors with poorly expressed EBV antigens [80, 81]. Due to the involvement of EBV in NPC, there is also the potential for the use of prophylactic and therapeutic vaccines for treatment. However, due to the diversity of EBVpathogenic mechanisms and EBV-related diseases, vaccines can only be designed for one disease entity, rather than all EBV-related malignancies [88]. Despite this shortcoming, a polyepitope-based vaccine has been developed for NPC that has numerous advantages over traditionally proposed vaccines that target EBV LMP antigens [89]. While still in the clinical stages of testing in humans, the vaccine has proven to be highly successful in mice populations.

Proteomics of HPV- and EBV-Associated Cancers Proteomics is a powerful approach for biomedical research because it aims for a comprehensive, quantitative analysis of protein expression and its changes under biological perturbations such as disease or drug treatment. Recent studies on stably transfected cancer (cervical and colon) cell lines have indicated that proteomics is powerful to identify the target proteins of E6 or E7 modulation as well as the E7-interacting proteins [90–92]. Analysis of the protein alterations and E7 binding partners in the transfected cells suggested that HPV-16 E7-infected epithelial cells could evade immune surveillance or resist against apoptosis by inducing or binding to chaperones, cell signaling and cellcycle regulatory proteins [90, 91]. Similar proteomic ­studies

J. Brumbaugh et al.

can be performed to unveil the target proteins and binding partners of E6 and E7 in HNSCC, which can provide further insight on mechanistic understanding of HPVs in head and neck oncogenesis and facilitate the development of anti-viral or anti-cancer drugs based on these target molecules and protein–protein interactions. The best-known cellular targets of the HPV-16 E7 oncoprotein are the retinoblastoma tumor suppressor protein pRB and the related pocket proteins p107 and p130. However, there is ample evidence that E7 has additional ­cellular targets that contribute to its transforming potential. To identify cellular targets of HPV-16 E7, tandem affinity purification can be used to pull down HPV-16 E7 associated cellular protein complexes and subsequently mass spectrometry (MS) can allow for the identification of cellular targets of E7. Using this approach, a 600-kDa retinoblastoma protein associated factor, p600, has been identified as a cellular ­target of E7. The protein regulates cellular pathways contributing to anchorage-independent growth and cellular transformation [93]. When applied to studying HPV-positive and HPV-negative cancers, proteomics could reveal target proteins that have diagnostic or therapeutic implication in the diseases [94–96]. For example, proteomics has been successfully used to identify a novel target protein, retinoblastoma-binding protein 48 (RbAp48), as an important mediator controlling the transforming activity of HPV-16 in cervical cancer. The protein was found differentially expressed between HPV-positive and HPV-negative cell lines and cancer tissues based on 2-D gel electrophoresis and MS. Suppression of RbAp48 using small interfering RNA in cervical epithelial cells significantly stimulated cell proliferation and colony formation. Conversely, over-expression of RbAp48 significantly inhibited cell growth and tumor formation [96]. These results suggest that proteomics profiling followed by molecular biology validation is a powerful approach to elucidate signaling molecules in HPV-associated cancers. Likewise, proteomics may have promising applications in EBV-positive NPC towards the mechanistic understanding of the disease and discovery of diagnostic/therapeutic targets [97]. Using proteomics and a phosphoprotein enrichment method, LMP1 was found to increase the quantity of total phosphoproteins by ~18%, and many proteins (e.g., annexin A2) showed significant changes in the degree of phosphorylation when LMP1 was expressed [98]. LMP1 increased the serine, but not tyrosine, phosphorylation of annexin A2 by activating the protein kinase C (PKC) signaling pathway [99]. EBV is able to efficiently immortalize primary B lymphocytes in  vitro. The growth program of EBV-infected B cells is initiated and maintained by the viral transcription factor EBNA2, which regulates viral and cellular genes, including the proto-oncogene c-Myc. Proteomic analysis has proven to be a powerful approach to profile the target proteins of EBNA2, including both c-Myc-dependent or

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c-Myc-independent ones [100]. EBV nuclear antigen leader protein (EBNA-LP) is a phosphoprotein suggested to play important roles in EBV-induced immortalization of B cells. One of the potential functions of EBNA-LP is a cooperative induction with EBNA-2 of viral and cellular gene expression, including that of the genes for viral LMP-1 and cellular cyclin D2. Based on MS analysis, the major phosphorylation sites of EBNA-LP were identified to be at serine residue of position 35 in the W2 repeat domain. These modification sites are critical for the protein to cooperate with EBNA-2 in upregulating the expression of LMP-1 in B-lymphoma cells [101].

levels, but few on transcriptomic and proteomic profiles [29,  102]. An improved mechanistic understanding of the ­virologic basis for HNCs would require profound analysis of these tumors using high-content molecular analysis technologies (e.g., proteomics). This would facilitate the development of targeted therapies for treatment of these cancers if immune escape can be reversed. Meanwhile, molecular classification of tumors is likely to provide important translational information that will allow a better estimate of prognosis and may well influence treatment decisions if future HPVstratified clinical trials support this approach.

Summary and Future Perspective

References

HPV infection has now been recognized as an important risk factor for a subset of HNSCC, particularly those arising from the oropharynx (base of tongue and tonsils). HPV-16 and HPV-18 represent the most prevalent viral types, and they show specific virus-tissue interactions in HNSCC. In addition, patients with HPV-positive HNSCC seem to have a better overall and disease-specific survival, as compared with the HPV-negative group. On the other hand, EBV has critical viral transforming functions in epithelial cells that may lead to the development of NPC, as evidenced by the consistent expression of EBV viral genes and latent membrane proteins in NPC. The tumorigenic activities of HPV in HNSCC and EBV in NPC have been well studied and documented, but there is a glaring lack of evidence regarding the interaction of HPV or EBV with precancerous lesions. Early diagnosis of virus-associated cancers is vital in combating the diseases, as it is much more effective when the tumors are treated at the early stage before metastatic spread. However, a positive detection of viral infection does not necessarily mean the development of cancer. In this regard, it is also important to develop new molecular biomarkers, in addition to viral DNA and proteins, for a more precise diagnosis of HPV- or EBV-associated HNSCC. Prophylactic vaccines have the potential to prevent and treat virus-associated HNC. However, it is equally important to develop molecular targeted therapies for patients with the cancers so as to slow down the progression of transformed cells and improve the survival. The mechanism of virus-associated tumorigenesis is complex, involving the aberrations of many signaling pathways and the alteration in expression of numerous proteins leading to immune escape by malignant cells. Although clinical studies have shown strong association between HPV/ EBV and subsets of HNCs, the molecular mechanism regarding how these viruses facilitate the development of HNC remains largely unclear. Previous molecular studies on HPV-associated HNCs have focused on DNA and chromosomal

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Chapter 8

Head and Neck Cancer Staging and Prognosis: Perspectives of the UICC and the AJCC Brian O’Sullivan and Jatin P. Shah

Abstract  The prognosis of head and neck cancer embodies numerous dimensions of outcome governed by a large array of factors within the patient and the tumor. These can be influenced by external factors that include access to an adequate standard of treatment for these tumors. For many outcomes, especially the key end-points of organ preservation, loco-regional control, occurrence of distant metastases, and survival, anatomic extent of disease remains one of the most powerful prognostic factors. This is embodied in the tumornode-metastasis (TNM) classification which has provided a very effective enabling tool to facilitate many elements of cancer control. Traditionally, its contribution has been a ­codified classification and language to describe anatomic stage of disease for use in the clinic, determining eligibility and stratification for clinical trials and treatment protocols, and for comparison and surveillance of treatment results among centers and jurisdictions. More recently, a focus on nonanatomic factors has become very important, partly because it is recognized that traditional extent of disease does not embrace all dimensions of prognosis. In particular, this relates to the quest to understand the biological dimensions of cancer that are needed to achieve more personalized and/ or biologically driven therapies. Increasingly, there is a need in head and neck cancer to exploit new biological discoveries to permit modification of treatment and interventions in the clinic for this heterogeneous group of tumors. Because of this, the TNM has been criticized due to a perception that it has not been adapted sufficiently to modern needs despite its worldwide adoption. This may stem from the fact that there is no alternative uniform functional framework available to classify nonanatomical predictive and prognostic factors. There seems to be a tendency to regard TNM as the optimal receptacle for these factors due to its uniform appeal and success. As the field evolves, both anatomic disease extent and other factors, especially those addressing biological B. O’Sullivan (*) Head and Neck Oncology Program, Department of Radiation Oncology, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, M5G 2M9, Canada e-mail: [email protected]

behavior of disease, need to be ­studied in their component domains as well as in combination using an agreed enabling taxonomy. An important strategy is to move toward constructing prognostic models, likely using prognostic nomograms, which will not only include the TNM staging information, but will also include other parameters of prognosis including comorbidities and biochemical or genetic markers. In addition, experts in one area (e.g., translational science or clinical trials methodology perhaps) who may rely on TNM may not always consider that the classification provides very different needs for others (e.g., health services research or screening and cancer control initiatives, etc.) and vice versa. Ignoring or dismissing one ­dimension of prognosis compared to another will not be fruitful and the true contribution of each will remain ­unappreciated, and the goals of the prognostic factor effort in head and neck cancer may be left unfulfilled. Keywords   Head and neck cancer • Staging • Prognosis • Prognostic models

Introduction In oncology, “to stage” a patient implies two intentions. The first uses clinical examination and investigations to describe the extent of disease to permit a rational treatment strategy to be formulated. The second employs an agreed classification system to categorize the extent of disease within risk hierarchies that predict the outcome following conventional treatment strategies. For the latter, the foremost priority is given to the risk of death and is provided by the joint primary tumor-node-metastasis (TNM) classification of the Union for International Cancer Control (UICC) and the American Joint Committee on Cancer (AJCC), a discussion about which will comprise much of this chapter. A challenge is to also consider new methods to enhance prognostic information and determine if these can be incorporated into or complement the traditional anatomically based classification. A variety of candidate areas exist and include features relevant to the host (or patient), the environment of the patient’s

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_8, © Springer Science+Business Media, LLC 2011

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treatment setting, and finally, the assessment of the tumor itself which has tended to receive the most emphasis. For the latter, of particular emphasis is the biological character of an individual tumor or groups of tumors. In this chapter, we discuss the importance of anatomic staging in the management of head and neck cancer and provide some perspective on the scope and application of the TNM classification and how it continues to evolve since its inception more than 50  years ago. A second component will summarize the changes that were introduced in the recently published seventh edition TNM [1, 2]. The final sections of the chapter address newer concepts including the evolving tension between anatomic staging in its current form and the value of nonanatomic methods of prognostication that need to be considered (see the section on “The Future of TNM in Head and Neck Cancer”).

Achievements, Challenges/Limitations, and Opportunities of the TNM Staging System 1. Anatomic extent of disease remains one of the most powerful prognostic factors and is embodied in the TNM classification. TNM has provided an effective enabling tool to facilitate many elements of cancer control on a global basis. Anatomic features of loco-regional tumor extension are especially important in the head and neck since these underpin the management of these tumors. 2. A major dilemma in TNM staging is that frequent revisions would undermine the value conferred by the stability and universality of TNM, but a static formulation of TNM risks falling behind the state of the art in diagnostic techniques, biological concepts, and biomarkers. 3. Dimensions of prognosis are not uniform and the settings where some factors are useful to consider may not apply to other situations (e.g., early vs. advanced stage, or recurrence vs. first presentation, or important end-point in head and neck cancer such as survival vs. organ preservation). 4. The TNM remains essential so that newer biological findings can be evaluated in the context of its existing structure. Although it has significant limitations in the era of molecular oncology, it is also needed to provide the framework for advances in biological discoveries when cohorts of patients are evaluated for prognostic or predictive outcomes. 5. Future research should focus on the evolution of biology with advancing stage since this could open the door to the potential for a true molecular-based “staging system.” A major achievement of this type (continued)

could override or complement traditional anatomic staging in some diseases or situations. 6. In considering prognosis in cancer, the UICC and AJCC are also focusing on host and environmental factors that may be as important as tumor-based prognostic factors in some settings. 7. The UICC and AJCC recognize an urgent need to achieve agreement on a new taxonomy and methodology to permit nonanatomic factors to be combined with traditional anatomic classifications while allowing the full impact of both to be explored, adopted, and used without compromise to the other. One ultimate goal is to move toward a prognostic nomogram, where the TNM anatomic staging will remain as an important component. 8. TNM serves many purposes in cancer care, research, and control, and dismissing one dimension compared to another will not be fruitful since the true contribution of each will remain unappreciated and the goals of the prognostic factor effort in head and neck cancer may be left unfulfilled.

The Principles of Staging in Head and Neck Cancer The Importance of Anatomic Staging in Head and Neck Cancer The challenge for oncologists who manage head and neck cancers is to achieve tumor control while maximizing the opportunities for preservation or restoration of form and function. A dominant pattern of treatment failure of head and neck tumors is loco-regional recurrence, making it important to have a clinical staging system that acknowledges this behavior and emphasizes the anatomic features of local tumor extension that underpin the management of these tumors. Clinical evaluation is a fundamental part of the assessment (i.e., palpation and observation of the head and neck that are almost unique to these sites because of their relative accessibility compared to other disease areas) and together with imaging studies informs a user friendly ­language for the extent of disease that can be applied uniformly and consistently on a worldwide basis [3]. This traditional need to classify the extent of disease remains a paramount component of the assessment of patients with head and neck cancer and the basis for many comparisons between groups of patients and the means to develop initial treatment approaches. Salvage of initial treatment failure also requires unique attention and diligence since selected patients may enjoy long-term control and cure if management is appropriately applied.

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Therefore, disease description at recurrence is important so that the goals of treatment are achieved and includes the ability to plan treatment and compile results that can be compared among centers and jurisdictions separately from the description of the initial treatment. Here again, a codified language to describe treatment and protocol guidelines and permit orderly reporting of results of this adverse setting is needed and is provided by an anatomic stage classification that is tailored to the recurrent scenario which in the TNM system uses the “r” prefix described later.

The Evolution of the TNM Classification in Head and Neck Cancer The TNM staging system was first proposed in 1944 by Pierre Denoix at Institute Gustave-Roussy, Paris, France [4]. The first formalization of the classification was developed by the Union for International Cancer Control (UICC) when it published the first of its brochures on cancer of the breast and larynx in 1958, to be followed by that on cancer of the buccal cavity and pharynx in 1963. This led to the classification of additional anatomic sites and their eventual compilation in 1968 as a single booklet, referred to as the Livre de Poche, which contained 22 body site classifications and represented the first edition of the TNM staging system [5]. Of central importance in the first edition of TNM were the classifications of head and neck ­cancer. These originally included buccal cavity, nasopharynx, hypopharynx, and larynx. All contained a common, though now outdated, regional lymph node classification that focused on whether lymph nodes in the neck were palpable or not, and used fixity as the criterion for N3. The buccal cavity was subdivided into seven regions and a number of subsites such as “lips (red borders)” with divisions into upper and lower components. Of interest also, the oropharynx was initially allocated as a region within the buccal cavity site and did not achieve independence as a region within the head and neck until the 1974 second edition [6]. Another interesting element was that fixation of the vocal cord was classified as T2 in the first edition and only became T3 in the 1974 second edition classification following a trial period of a new proposal. Also the first edition contained only a limited attempt to combine the three different anatomic components (T–N–M) into groups that might provide prognostic strata as stage-groups. This process was confined to breast and cervix cancer as it was deemed “…in the opinion of the Union an attempt to stage group all sites would at present be immature” [5]. Importantly, this was also modified in the second edition thereby representing the first formal international attempt to prognosticate in head and neck cancer using different elements of extent of disease grouped together. The American Joint Committee (AJC) was founded in 1959 to complement this work in the USA. Joint classifications

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were prepared by both organizations and distributed for trial periods before their formal adoption into the TNM classification. In 1977, the AJC introduced a TNM classification of its own [1] which had the potential for two separate classifications. This was recognized early on and a strong collaboration between both organizations (the AJCC renamed in 1980, and UICC) has continued since, so that both classifications resemble each other as closely as possible. Nowhere is this more apparent than in the classification of the head and neck sites stewarded by the authors of this chapter representing the UICC and the AJCC. From the outset, the TNM was intended to be an anatomic stage classification describing the anatomic extent of the primary tumor as well as the involvement and extent of regional lymph nodes and distant metastasis. It describes the anatomic extent of cancer and is based on the hypothesis that the probability of survival and the choice of treatment are related to the anatomical extent of the tumor at the primary site (T), the presence or absence of tumor in regional lymph nodes (N), and the presence or absence of metastasis beyond the regional lymph nodes (M). At present, in the head and neck sites, T is almost always divided into four major categories (T1–T4), with a further subdivision into moderately advanced local disease (T4a) or very advanced local disease (T4b). A common lymph node classification represented by four categories (N0–N3) with some subcategories is used in almost all the head and neck sites. The T- and N-categories are also combined with the M-categories that indicate the presence or absence of distant metastases to form groups representing stages and that confer prognostic guidance. As noted earlier and continues to be the case, TNM has always needed to evolve with the availability of additional information about outcome, new treatments, or novel ways to evaluate disease and anatomy, including developments in imaging or emerging biological insights about disease behavior or etiology. Almost all clinical trials use anatomic extent, generally represented by the TNM or its elements, to define entry criteria or to control for prognostic imbalance between arms of randomized trials by employing stratification based on anatomic stage [7]. It is also a critical pathway to developing clinical practice guidelines such as those of the National Comprehensive Cancer Network (NCCN) [8], and is a key determinant in identifying patients to be treated by guidelines, and for monitoring compliance to guidelines [7].

The Place of Non-anatomic Prognostic Factors and Staging It is important to recognize that the TNM classification was never intended to capture all elements that are important in determining prognosis or guiding treatment and that a variety of tumor, host, and external factors are also important and

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are becoming increasingly so today. One of the ironies of the TNM classification is that it has been immeasurably successful in its goals and has enjoyed worldwide adoption but in recent times has become a target for criticism because of assertions that it has not adapted itself to modern needs [9]. This may stem from the fact that there is no uniform functional framework that can be used to classify nonanatomical predictive and prognostic factors. The tendency seems to have evolved to consider the TNM as the optimal receptacle for these factors presumably due its uniform appeal and success. This needs to be considered carefully since the problem is not straightforward. Dimensions of the elements of prognosis are not uniform and the settings where some ­factors are appropriate to consider may not apply to other situations of the disease. These concepts will be discussed later.

How TNM is Modified As discussed already, changes continually take place in the TNM classification because of the need to maintain relevance with current management approaches and to respond to the availability of new data that may be considered in revisions to the classifications. This generally requires evidence of the need for modification and for the most part relies on published data in the literature. Thus, for example, the AJCC and UICC meticulously reviewed the overall TNM classification for all diseases in preparation for the recently published seventh edition. In considering change, it is important to reflect on the fact that any classification or staging system is a “compromise” between the “ideal” and the “practical.” The more complex the system is, the less compliance we will observe. One of the basic tenets of the staging system is that it should be applicable and available worldwide, it should be user friendly, and it should have the ease to have maximum compliance from all parts of the world [10]. The process of revision involves collaboration between both organizations, and that is partly accomplished by a series of disease-specific task forces. A number of resources are available to the task forces which especially include a structured process for introducing changes to the TNM classification. The elements of the TNM process include the development of unambiguous criteria for the information and documentation required to consider changes in the classification, establishment of a well-defined process for the annual review of relevant literature, formation of site-specific expert panels, and the participation of experts from all over the world in the TNM review process [11]. In the preparation of the seventh edition, a number of anatomic areas were considered by the head and neck task forces and are summarized later (see the section on “Recent Modifications to TNM”). In addition some domains, including anatomically based issues, may seem relevant but are not included in

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the modifications. This may arise because the data supporting the change are not sufficiently strong, or may lack the practicalities to permit its inclusion in a general way, or may not fit into the established structure of the TNM. In order to address the need for awareness of other elements that are not included in the formal classification, the UICC and the AJCC have initiated separate processes with different but complementary goals. The UICC approach includes a separate publication, entitled the “TNM Supplement, A Commentary on Uniform Use” [12]. The “Supplement” now appears following each revision of TNM with the fourth edition currently in preparation. Its purpose is to provide explanations and examples to answer the numerous questions that arise during the daily use of TNM, particularly in unusual cases. It enumerates the recommended criteria for pathological classification (pT and pN). One example in the head and neck is a description of the superior and inferior boundaries of the glottis, since these are not elaborated in the UICC Livre de Poche though such items may be included in the more expansive AJCC Cancer staging manual. Another example concerns the reminder that pathological classification also uses clinical information. Thus in considering impaired mobility or fixation in the glottis, this information that is evaluated in the clinical T-category is also used to define the pathologic TNM (see Table 8.1) [12]. The “Supplement” also contains proposed classifications for new tumor sites and types not yet part of the official UICC and AJCC TNM system and that can be tested by interested investigators with a view to encouraging publication that may result in their subsequent inclusion in the formal classification if the data prove robust. Optional expansions of existing TNM categories are also included in the “Supplement” for those needing to record more detail. An added feature is the “Frequently Asked Questions” chapter, derived from the UICC and AJCC TNM web sites’ Help Desks. The AJCC has taken a different approach. First, the AJCC staging manual is a more expansive text. Consequently, it is less portable for consultation in the clinic by clinicians, though it provides the reference foundation for the work of cancer registrars in North America. A more compact version is available though is still not as brief and synoptic in presentation as the UICC Livre de Poche. In addition, the AJCC has implemented the “Collaborative Staging System” (CS), which acts as a repository of all available prognostic information for current and future use. This process commenced in 2004, and comprises a data collection tool across all US hospital and population registries for cancer staging information [13]. It uses a standardized data dictionary to collect information on T, N, M, and site-specific prognostic and predictive factors. The CS system is built into all cancer registry software systems in the USA. Areas identified for data collection in the head and neck sites include such factors as the

8  Head and Neck Cancer Staging and Prognosis Table 8.1  Application of selected rules relevant to the TNM head and neck classification General issues For each disease, there should be a clinical (obtained without resection) and a pathological (obtained after surgery) classification that contain equivalent descriptors Pathological classification (pTNM) is based on evidence acquired before treatment, supplemented or modified by additional evidence acquired from surgery and from pathological examination Because the designation is based on evidence acquired before treatment, a glottic cancer with a fixed vocal cord will remain a T3 lesion after surgery unless additional evidence of extension of disease is present to raise the category to the next (i.e., more advanced) level The pathological assessment of pT and pN requires a resection adequate to evaluate the highest pT or pN category If there is doubt about whether a tumor should be classified with a higher T- or N-category, it should be allotted to the lower category (i.e., less advanced) where the available criteria for that case can be reliably applied The designation X is used for the T- or the N-categories, if there is inadequate information available to classify the lowest category when disease has been known to be present in that location. The term X is not used for the M-category since a clinical exam alone can permit assessment of distant metastases. It is also not used for the designation of unknown primary where T0 is the correct convention T-category issues Tumors overlapping adjacent areas should be classified according to the site where the bulk of the lesion is located In the case of multiple primary tumors in one organ, the tumor with the highest T-category should be classified and the multiplicity or the number of tumors should be indicated in parenthesis, e.g., T2(m) or T2(5) In simultaneous bilateral primary cancers of paired sites (e.g., tonsillar carcinomas), each tumor should be classified independently In unknown primary cancer classification, the designation T0 should be used for the T-category. T0 is also used at the time of recurrence of a previous known head and neck cancer (e.g., regional lymph node or distant failure) if there is no evidence of disease recurrence at the primary site, preceded by the descriptor “r” N-category issues The regional lymph nodes are the cervical nodes. Midline nodes are considered ipsilateral nodes except in the thyroid The definitions of the N-categories for all head and neck sites except nasopharynx, thyroid, and mucosal melanoma are the same In oral cavity, larynx, pharynx, and thyroid cancers, metastases at level VII (those in the anterior superior mediastinum, cephalad to the innominate artery) are considered regional lymph node metastases. The remaining mediastinal lymph node metastases are considered distant metastases Histological examination of a selective neck dissection specimen will ordinarily include 6 or more lymph nodes Histological examination of a radical or modified radical neck dissection specimen will ordinarily include 10 or more lymph nodes If the lymph nodes are negative, but the number ordinarily examined is not met, classify as pN0 When size is a criterion for pN classification, measurement is made of the metastasis, not of the entire lymph node In unknown primary cancer classification, the designation T0 and the N-classification should use that of the site most likely to represent the origin of the tumor

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actual size of lymph nodes, the location of lymph nodes (e.g., upper or lower neck involvement), human papillomavirus (HPV) status, the presence of extracapsular spread (ECS), and tumor thickness in oral cancers. Many of these are not reliably available by clinical evaluation, but their strength is apparent on pathological examination where they may influence practice in significant ways. For example, the presence of ECS is a singularly adverse factor [14] and drives the need for chemotherapy in addition to radiotherapy in the postoperative adjuvant management of cervical lymph node metastases [15]. Tumor thickness in oral cavity primary sites is one of the strongest predictors for the presence of lymph node involvement in the neck beyond the formal T staging system [16], thereby influencing the approach to neck management. Other important pathological issues that are not part of the TNM at present include the character of the tumor (e.g., endophytic vs. exophytic) and the nature of the host tumor interface (pushing vs. infiltrating) and the presence of perineural or lymphovascular invasion (LVI) that also impact on the treatment of patients. In addition to being implemented in some other jurisdictions beyond the USA, ongoing efforts involving the College of American Pathologists (CAP) and the Centers for Disease Control and Prevention (CDC) are revising the CAP Cancer Templates for reporting pathology on cancer specimens to collect core elements on tumor size, extension, nodal involvement, and metastases in the format needed for recording in the CS system. It is also expected that the CS system will be incorporated in the NCI’s Cancer Bioinformatics Grid (caBIG) as the accepted standard for recording data on the extent of disease and stage [13]. In this way, the future potential exists for important elements that influence treatment and prognosis to be analyzed in order to develop prognostic groups that may be able to enhance the existing TNM stage classification.

Specific Designations and Rules in TNM The staging of head and neck cancer requires the clinician and the cancer registrar to be familiar with an extensive assortment of anatomic sites and subsites. Practitioners and statisticians interested in how results from clinical trials are interpreted and received need to be familiar with the fundamental rules of the TNM classification. The same holds for everyone involved in interpreting and applying the general results of treatment or in maintaining and addressing consistency in how treatment guidelines are developed, used, and assessed. Depending on an individual’s or a group’s focus, some of these may seem arbitrary, cumbersome, or even unnecessary. Nonetheless, they embody a uniformity that is applicable to all oncologic disease sites, health professionals, and jurisdictions around the world [3].

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A detailed discussion of the rules of TNM is not intended in this chapter. Some basic issues will be known to practitioners such as the fact that the TNM for most mucosal sites is designed for squamous cell carcinoma and minor salivary gland cancer. It is also acknowledged that head and neck oncologists are very familiar with the TNM system though they may not be aware of some of the recent changes described below. In addition, even experts may not be aware of all of the “fine print” that exists and a summary of some of the questions and problems that arise in day-to-day usage is provided (see Table 8.1). This is not intended to be exhaustive and the interested specialist should also consult additional sources mentioned earlier as well as the actual TNM classification publications [1, 2, 12]. Several broader issues merit comment, however. These concern the areas of clinical vs. pathological staging, some additional descriptors within the classification, and the use of grouping of elements to define prognosis.

Clinical vs. Pathological Staging All cases should be confirmed microscopically and two classifications are described for each site, which includes a Clinical classification (the TNM or cTNM) that is based on evidence acquired before treatment and is essential to select and evaluate therapy. Physical examination, imaging, endoscopy, biopsy, and other relevant examinations including surgical exploration comprise the majority of this evidence. In contrast, Pathological classification (pTNM) is based on postsurgical histopathological classification, and is used to guide adjuvant therapy and provides additional data to estimate prognosis and to calculate end results. Both should be recorded and should not be mixed or considered equivalent since different selection criteria apply to each. In addition, they should contain the same elements.

Additional Descriptors Used in TNM The clinical TNM and pTNM classification also contain specific terms to facilitate clinical situations faced by clinicians in the contemporary management of head and neck cancer. Thus, several symbols may be used to facilitate including the m, y, r, and R identifiers (see Table 8.2). The suffix m, in parentheses, is used to indicate the presence of multiple primary tumors in a single site, whereby the tumor with the highest T-category should be classified and the multiplicity or the number of tumors should be indicated in parenthesis, e.g., T2(m) or T2(2) in the case of two tumors (see Table 8.1).

B. O’Sullivan and J.P. Shah Table 8.2  Selected additional descriptors encountered in the TNM or pTNM of head and neck cancer The suffix m, in parentheses, is used to indicate m Symbol the presence of multiple primary tumors at a single site. See commentary in Table 8.1 y Symbol In those cases in which classification is performed during or following multimodality therapy, the cTNM or pTNM category is identified by a y prefix The ycTNM or ypTNM categorizes the extent of tumor actually present at the time of that examination. The y categorization is not an estimate of the extent of tumor prior to multimodality therapy This convention should typically be used following neoadjuvant therapies and may be most applicable to induction chemotherapy r Symbol Recurrent tumors, when classified after a disease-free interval, are identified by the prefix r R-Classification The absence or presence of residual tumor after treatment is described by the symbol R as follows RX: Presence of residual tumor cannot be assessed R0: No residual tumor R1: Microscopic residual tumor R2: Macroscopic residual tumor In some situations, the R2 designation may interact with the “r symbol” if macroscopic (gross) residual represents recurrence of previous tumor (see text)

The y symbol is available to classify cases during or following multimodality therapy by identifying the clinical TNM or pTNM category identified by a “y” prefix that designates that the classification refers to the extent of tumor actually present at the time of that examination. Therefore, the y categorization is not an estimate of the extent of tumor prior to multimodality therapy, but is useful for description of TNM after the completion of neoadjuvant regimens [17]. The lower case “r” symbol is available to describe recurrent tumors and needs to be applied after a disease-free ­interval (usually in the order of 3–4 months). Such tumors are identified by the prefix “r” as rTNM or rpTNM and need to be distinguished from the upper case “R” designation used to describe residual disease following surgical resection as R0 for microscopically clear resections, R1 for microscopic residual disease, and R2 for macroscopic residuum. In some cases, confusion could arise between the upper case “R2” designation for gross residual disease vs. the lower case “r” designation that designates recurrent disease since one may eventually merge into the other if sufficient time evolves. This is especially prone during the time to referral to a cancer center for definitive treatment following an initially incomplete excision.

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Lymph Node Classification for Micrometastasis and Sentinel Node Assessment The regional lymph node classification has recently also been adapted to address subclinical disease. This is particularly relevant in the head and neck to sentinel lymph node assessment where the designation “Sn” has been introduced in the TNM classification (Table 8.3). Therefore, the following designations are applicable when sentinel lymph node assessment is attempted: pNX(sn), sentinel lymph node could not be assessed; pN0(sn), no sentinel lymph node metastasis; and pN1(sn), sentinel lymph node metastasis. Cases with morphological evidence of micrometastasis only, i.e., no metastasis larger than 0.2 cm, can be identified by the addition of “(mi),” e.g., pN1(mi) (see Fig. 8.1). A designation of morphologically evident isolated tumor cells (ITC) can also be used to designate single tumor cells or small clusters of cells not more than 0.2 mm in greatest extent that can be detected

Table  8.3  Refinement in description of subclinical disease (most applicable to regional lymph node evaluation using sentinel node biopsy) assessment Cases with micrometastasis only, i.e., no metastasis larger than 0.2 cm, can be identified by the addition of (mi), e.g., pN1 (mi) Isolated tumor cells (ITC) are single tumor cells or small clusters of cells not more than 0.2 mm in greatest extent are designated by the term “i+” Molecular detection (nonmorphologic findings for ITC) of tumor presence is designated by the term “mol+” Sentinel node assessment is described by the use of the suffix “sn” at the end of the classification of a given tumor as depicted below The classifications for ITC and molecular detection of tumor should be used and designated as follows pN0 No regional lymph node metastasis histologically; no examination for ITC pN0(i−) No regional lymph node metastasis histologically; negative morphological findings for ITC pN0(i+) No regional lymph node metastasis histologically; positive morphological findings for ITC pN0(mol−) No regional lymph node metastasis histologically; negative nonmorphological findings for ITC pN0(mol+) No regional lymph node metastasis histologically; positive nonmorphologic findings for ITC

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by routine H and E stains or immunohistochemistry and is designated as (i+) (see Table 8.3). This overall approach has been validated recently by experts in sentinel lymph node assessment [18]. The approach has been similarly adapted to the situation where no morphological evidence of disease is apparent, but evaluation is based on a molecular assessment of the presence of disease by techniques such as flow cytometry or DNA analysis (see Table 8.3). The term “mol” is used to indicate that such a technique has been employed in the assessment; e.g., pN0(mol−) indicates that no regional lymph node metastasis is present histologically, and there is a negative assessment for nonmorphological findings for ITC. In contrast, pN0(mol+) indicates that no regional lymph node metastasis is identifiable histologically but there is a positive assessment for nonmorphological findings for ITC. Also, in the situation where these characteristics have been assessed but confined to a sentinel lymph node assessment, the term “Sn” may be used as follows: pN0(mol+)(sn), no sentinel lymph node metastasis histologically, but there are positive nonmorphological findings for ITC. In general, these terms are not commonly used in practice, but are available in the event that these assessments become more uniformly used in the future. It is apparent that the designations (i+) and (mol+) are considered N0 at this time.

Stage Grouping For purposes of tabulation and analysis, it is useful to condense the T-, N-, and M-categories into stage groups. In general, in the TNM system, the groups are based on a hierarchy governed by the degrees of modification of prognosis. Most usually carcinoma in situ is categorized as Stage 0, tumors

When sentinel lymph node assessment is attempted pNX(sn) Sentinel lymph node could not be assessed pN0(sn) No sentinel lymph node metastasis pN1(sn) Sentinel lymph node metastasis Cases with or examined for ITC in sentinel lymph nodes can be classified as follows pN0(i−)(sn) No sentinel lymph node metastasis histologically, negative morphological findings for ITC pN0(i+)(sn) No sentinel lymph node metastasis histologically, positive morphological findings for ITC pN0(mol−)(sn) No sentinel lymph node metastasis histologically, negative nonmorphological findings for ITC pN0(mol+)(sn) No sentinel lymph node metastasis histologically, positive nonmorphological findings for ITC

Fig. 8.1  Micrometastasis evident by small clusters of cells not more than 0.2 mm in greatest extent can be detected by routine H and E stains and are designated by the addition of “mi,” e.g., pN1(mi) for detection in a single lymph node. Single tumor cell can also be classified using the term isolated tumor cells (ITC) and designated by the use of (i+) (see Table 8.3)

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localized to the organ of origin as Stages I and II, locally extensive disease and especially spread to regional lymph nodes as Stage III, and those with distant metastasis as Stage IV. In the classification of head and neck tumors, some unique differences exist and will be outlined in the sections that address specific anatomic sites in the head and neck region, most notably in the area of mucosal melanoma, where a new classification has been introduced for the first time, in anaplastic thyroid cancer and in the general head and neck classification where very advanced local disease (T4b) and extensive regional adenopathy (N2c and N3) will place the case at the highest level of adverse prognosis (Stage IV). The stage groups are intended, as far as possible, to provide homogeneous groups with distinctive survival rates for the different cancer sites. In addition, there are pathological stage groups if sufficient tissue has been removed for pathological examination to evaluate the highest T- and N-categories. As discussed earlier, the Stage Groups have also evolved over time. Originally, in the first edition of the TNM classification, they did not exist and in the most recent edition, the AJCC and the UICC have introduced separate modified approaches in order to acknowledge the potential importance of nonanatomic factors (see “Combining Variables and Validation” section later).

Recent Modifications to “TNM” The seventh edition of the TNM staging system has recently become available for wide usage [1, 2]. In the head and neck classifications, some additional fine tuning has been undertaken but significant changes were not necessary following the relatively substantial modifications previously introduced in the sixth edition [19, 20]. These had included similar T-category criteria for primary tumors of the oral cavity, oropharynx, salivary glands, and thyroid gland. However, some adjustments seemed necessary and are briefly outlined in the following paragraphs. In addition, there is one new classification that addresses mucosal melanoma of the head and neck. Broadly speaking, the changes are intended to reflect current practices of treatment, clinical relevance, and contemporary data as well as providing the opportunity for data to be collected with a uniform classification in situations where this may have been problematic previously.

Recent Modifications to the T-Classification Very Advanced Local Disease (T4) In the seventh edition, the terms “resectable” (T4a) and “unresectable” (T4b) that were introduced by the AJCC in the sixth edition [19], have been replaced by the words “moderately

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advanced” (T4a) and “very advanced” (T4b). These changes were considered necessary since a significant proportion of advanced stage epithelial malignancies of the head and neck are being treated nonsurgically, and hence the terms “resectable” and “unresectable” were felt to be inappropriate for the selection of therapy. Moreover, criteria for resectability may be subjective and are often dependent on the quality of available imaging studies [21] that may not be universally available across the world. In addition, authors have reported that in some sites certain T4b cancers may be resectable with a favorable outcome, raising concern that the criteria may not be universally applied especially in retrospective series [22]. The anatomic criteria for the definitions of T4a and T4b, however, remain unchanged. For oropharynx, hypopharynx, larynx, nasal cavity, paranasal sinuses, and major salivary glands, T4 lesions have been divided into T4a (moderately advanced local disease) and T4b (very advanced local disease) leading to the stratification of Stage IV into Stage IVA (moderately advanced local/regional disease), Stage IVB (very advanced local/regional disease), and Stage IVC (distant metastatic disease). An exception is the nasopharynx. Here there is as yet insufficient data to permit a subdivision of the T4 category. In particular, there is evidence that minimal invasion of the skull base or minimal cranial nerve involvement is not uniformly prognostically detrimental when determined by imaging assessments [23], further emphasizing the rationale for the importance of clinical evaluation in staging assessments (e.g., of cranial nerves in this instance). Further information is needed to explore the cutpoints of this heterogeneous T-category in addressing further revisions to the nasopharyngeal carcinoma (NPC) TNM.

Nonmelanoma Skin of the Head and Neck Changes have also been introduced in the T-categories of nonmelanoma skin cancers that are also consistent with other cutaneous sites in the body. The 5 cm size breakpoint between the T2 and the T3 categories and invasion of extradermal structures as a criterion for T4 have been eliminated. Instead, a more clinically applicable approach for T3 includes invasion of peripheral facial and skull bones more readily amenable to safe ablative approaches, whereas the T4 category is confined to involvement of the skull base. Skin lesions in non-head and neck sites are similarly categorized according to relevant anatomic issues (e.g., axial skeleton in T4). Another novel feature is T1 tumors (<2 cm in size) can be upstaged to Stage II if they contain two or more defined high-risk criteria that include some pathologic features and location of the lesion, though in practice this will only apply to a small number of cases. The latter approach, using such high-risk features that contain pathological rather than anatomic factors, is unique to the AJCC version of the classification, but are still specified separately by the UICC without formally impacting the classification.

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Other Changes in T-Categories Relatively minor changes have been made in the remaining T-categories for various primary sites in the head and neck. The most apparent changes are in the nasopharynx (see Table 8.4), a site that underwent no change in the sixth edition TNM other than some minor wording. However, since the sixth edition, a relatively consistent finding has been the absence of a difference in outcome between T1 and T2a tumors leading to a recommendation for reclassification of patients with soft tissue disease involvement of the oropharynx and nasal fossa to the T1 category [24, 25]. Thus, T2a lesions will now be designated T1 and Stage IIA will therefore now be Stage I (see Table 8.4). Table  8.4  Nasopharyngeal TNM clinical classification (revision in seventh edition) T – primary tumor TX Primary tumor cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ T1 Tumor confined to nasopharynx or extends to oropharynx and/or nasal cavity T2 Tumor with parapharyngeal extensiona T3 Tumor invades bony structures of skull base and/or paranasal sinuses T4 Tumor with intracranial extension and/or involvement of cranial nerves, hypopharynx, orbit, or with extension to the infratemporal fossa/masticator space N – regional lymph nodes NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Unilateral metastasis, in cervical lymph node(s), and/or unilateral or bilateral metastasis in retropharyngeal lymph nodes, 6 cm or less in greatest dimension, above the supraclavicular fossa N2 Bilateral metastasis in lymph node(s), 6 cm or less in greatest dimension, above the supraclavicular fossa N3 Metastasis in lymph node(s) greater than 6 cm in dimension or in the supraclavicular fossa N3a greater than 6 cm dimension N3b in the supraclavicular fossa M – distant metastasis M0 No distant metastasis M1 Distant metastasis Stage grouping (Nasopharynx) Stage 0 Tis N0 M0 Stage I T1 N0 M0 Stage II T1 N1 M0 T2 N0, N1 M0 Stage III T1, T2 N2 M0 T3 N0, N1, N2 M0 Stage IVA T4 N0, N1, N2 M0 Stage IVB Any T N3 M0 Stage IVC Any T Any N M1 Note: The term “Stage Grouping” is termed “Anatomic Stage/Prognostic Groups” in the AJCC version of the classification [1] a  Parapharyngeal extension denotes postero-lateral infiltration of tumor Adapted from Sobin LH, Gospodarowicz MK, Wittekind C. TNM classification of malignant tumors. 7th ed. New York: Wiley; 2010. Reprinted with kind permission from Wiley

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As well, in the substantial series from Hong Kong that influenced the preceding recommendation concerning the T2a category, 1006 patients had T2b disease (those with parapharyngeal extension). Analysis of this subset indicated that they remain a distinct group with less favorable prognosis compared to the proposed T1 category. A significantly higher hazard of local and distant failure is evident with consequent significant impact on cancer-specific death. This effect was even more apparent when restricted to patients without lymph node involvement [24] and was confirmed in a separate large series from mainland China that included 309 patients with T2b disease [25]. Both series demonstrate a more even rise in the hazard ratio for adverse events with redistributed T-categories (i.e., T2a is reassigned to T1, and T2b remains as a T2 category). Therefore, this is adopted in the seventh edition TNM that includes the subcategory characterized by the presence of parapharyngeal extension without additional extension representing the T2 category alone. As a consequence, former Stage IIB of the sixth edition is now designated Stage II in the seventh edition (see Table 8.4). For primary cancers of the thyroid gland, T1 tumors have been subdivided into T1a (£1  cm) and T1b (>1  cm, up to 2 cm), limited to the thyroid gland. This subdivision of T1 will allow gathering of data to clarify the much debated controversy in the literature about the management of primary tumors <1 cm (microcarcinoma) and >1 cm, up to 2 cm confined to the thyroid gland. In the formal TNM classification, both the AJCC and the UICC recommends to continuing the use of traditional descriptors of multifocality using the designation (m) (the largest determining of the classification), e.g., T2(m). In this context, the AJCC has also introduced a descriptor for solitary tumor (s) in addition to multifocal tumor (m) to permit users to subdivide the T-categories.

Recent Modifications to the N-Classification Traditionally, the N-classification for cervical lymph node metastasis has been uniform for all sites except thyroid, nasopharynx, and skin. The N-classification for thyroid and nasopharynx is unique to those sites and is based on tumor behavior and prognosis. In NPC, retropharyngeal lymph nodes, regardless of unilateral or bilateral location, will now be considered N1. The basis for this decision reflects several issues. First, heterogeneous approaches to addressing retropharyngeal nodes have been used among centers. Some have considered them as N1 if unilateral, N2 if bilateral, N1 irrespective of laterality, N1 if discrete, or T2b if abutting adjacent soft tissue tissues or unclassified [24]. Some of these approaches reflect historic inadequacies in imaging prior to the era of cross-sectional imaging, especially MRI, and consistent principles have not been identified in TNM. Evidence from several studies shows that patients with retropharyngeal nodes alone have a risk of distant metastasis that is similar to

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N1 disease [26, 27]. In addition, the proposal that they should correspondingly be classified as N1 disease and that this should be independent of laterality [27] forms the basis for revision of this element in the seventh edition of TNM. An important change for nonmelanoma skin cancer in the seventh edition is the introduction of the N-classification used in the remaining head and neck sites and is justified based on a variety of studies that indicate that increasing extent of neck disease is associated with adverse outcome [28]. Indeed this compelling argument has influenced the complete nonmelanoma skin cancer classification to a degree that the head and neck N-classification is now also used for axillary and inguinal lymph nodes in the seventh edition TNM. For metastatic squamous cell carcinoma, from mucosal primary sites, no major changes were made in the N staging for any site, except that a descriptor has been added. As noted earlier, ECS of disease has been added as ECS+ or ECS− as a descriptor for capture in the CS of the AJCC. These descriptors will not influence the nodal staging system, but will permit gathering of data for potential future revisions of the N-classification. A final point concerning the neck is that the new classification for mucosal melanoma (see below) uses a limited schema restricted to only designating absence (N0) or presence of regional lymph node involvement (N1) without additional categories (see Table 8.5). Table 8.5  TNM classification for mucosal melanoma of the head and neck (a new classification in the seventh edition TNM) Primary tumor T3 Mucosal disease T4a Moderately advanced disease Tumor involving deep soft tissue, cartilage, bone, or overlying skin T4b Very advanced disease Tumor involving brain, dura, skull base, lower cranial nerves (IX, X, XI, XII), masticator space, carotid artery, prevertebral space, or mediastinal structures Regional lymph nodes NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastases N1 Regional lymph node metastases present Distant metastasis M0 No distant metastasis M1 Distant metastasis present Stage grouping Stage III Stage IVA

T3 N0 M0 T4a N0 M0 T3–T4a N1 M0 Stage IVB T4b Any N M0 Stage IVC Any T Any N M1 Note: The term “Stage Grouping” is termed “Anatomic Stage/Prognostic Groups” in the AJCC version of the classification [1] Adapted from Sobin LH, Gospodarowicz MK, Wittekind C. TNM classification of malignant tumors. 7th ed. New York: Wiley; 2010. Reprinted with kind permission from Wiley

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The New Classification for Mucosal Melanoma of the Head and Neck Mucosal melanoma of the head and neck warrants separate consideration and the approach to these lesions is outlined in a new chapter that introduces a TNM classification for the first time (see Table 8.5). Even small cancers behave aggressively with high rates of recurrence and death [29]. To reflect this aggressive behavior, even the smallest mucosal melanomas confined to the mucosa alone are designated as T3 and those with moderately advanced lesions (involving underlying cartilage or bone) are staged T4a. Very advanced primary tumors are staged T4b. In situ mucosal melanomas are excluded from staging, as they are extremely rare. There is also no T1 or T2 category. It is intended that the availability of a stage classification for this rare, unfavorable, and perplexing disease may facilitate research addressing its etiology, biology, and treatment.

The Future of TNM in Head and Neck Cancer As implied and discussed earlier, the anatomic extent of disease remains one of the strongest and most consistent prognostic factors, especially in head and neck cancer. Multiple reasons for this exist and have been described. As also mentioned, however, its very success seems to have rendered it vulnerable since no alternative overarching strategy has emerged to amalgamate, administer, and process multiple prognostic elements for a given cancer. A major dilemma in TNM staging is that frequent revisions to include new biomarkers, for example, would undermine the value conferred by the stability and universality of TNM, but a static formulation of TNM risks falling behind the state of the art in diagnostic techniques, biological concepts, and biomarkers [30]. In fact, other techniques do exist and should be considered but a shift in attitude is probably needed to embrace other methods of classification in addition to the TNM system. In addition to the area of biomarker discovery, other areas of prognostic importance also exist and in many situations have the capability of equaling or even overcoming effects embodied by traditional areas of cancer classification in terms of disease biology and anatomic disease extent. For example, many nonanatomic ­factors address issues relevant to the host (i.e., patient) or the environment or setting where the patient is treated and particularly in the context of the availability of treatment or diagnostic assessments, but receive scant attention in the voluminous literature on prognosis that has emerged recently. Some of these issues will be discussed to introduce these concepts while recognizing that this field is evolving

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and immediate solutions have not yet been developed or universally adopted. Broadly, prognostication in cancer can be classified into three domains that address the dimensions of the Tumor, the Host, and the Environment. This traditional classification has been used by the UICC in its publications “Prognostic Factors in Cancer” now in its third edition [31]. In addition, this text has also introduced a tabular format for each disease site throughout the body to address these three dimensions but, additionally, has allocated them into three hierarchy tiers to address whether these factors influence treatment of the disease at the present time (based on recommendations in published practice guidelines), whether they add valuable additional information to understand the disease setting without influencing treatment decisions, or finally whether they represent new and promising discoveries that have not yet found a place to put it in the assessment of the disease in the clinic. A modified example of one of the head and neck tabulations is shown in Table 8.6 [32]. Some of these areas will be discussed briefly in addition to some of the challenges in grouping data and using them to Table 8.6  Prognostic factors in oral cavity, pharynx, and larynx cancer Prognostic Environment factors Tumor related Host related related Essential T-category Performance status N-category M-category Anatomic subsite Additional

Resection margin Number of involved nodes Extracapsular nodal extension Perineural, lymphovascular invasion Tumor hypoxia HPV status

New and promising

EGFR expression Surgical molecular margins Osteopontin DNA profiling

Comorbidities Age

Radiation dose Overall treatment time Quality of surgery and radiotherapy

Sources: ESMO guidelines for management of SCC of the head and neck 2005 http://www.esmo.org/reference/referenceGuidelines/pdf/new_pdf/ ESMO_16_SCCHN.pdf National Cancer Institute: Lip and Oral Cavity (PDQ®): Treatment Guidelines 2005 http://www.cancer.gov/cancertopics/pdq/treatment/ lip-and-oral-cavity/healthprofessional/NCCN Clinical Practice Guidelines in Oncology: Head and Neck Cancer 2005 http://www.nccn. org/professionals/physician_gls/PDF/head-and-neck.pdf Modified from Bourhis J. Oral cavity, pharynx, and larynx cancer. In: Gospodarowicz MK, O’Sullivan B, Sobin LH, eds. Prognostic Factors in Cancer. 3rd ed. New York: Wiley; 2006:99–104

prognosticate for the individual patient or in groups of patients. In addition, statistical assessments need ongoing understanding of concepts that address validation in particular.

The Importance of  “Non-anatomic” Tumor Factors Introduction of Biologic Prognostic Markers A recent provocative editorial [33] noted that the power of the TNM staging system is largely derived from the observation that tumors demonstrating loco-regional or distant spread carry a worse prognosis than their less advanced counterparts. The problem is that, while this is true, and it is possible to predict survival based on a particular clinicopathological stage, there are clearly some patients that beat the odds [33]. Unfortunately, the authors also point out that there is also evidence that small tumors can metastasize early in their course and that a surgically resected primary tumor may in fact harbor cells demonstrating metastatic potential. This suggests the possibility to differentiate virulent tumor cells capable of metastasis from nonvirulent tumor cells based on molecular profiling. Molecular evidence may then be used to predict the outcome and treatment needs for an individual patient better than TNM staging. This speaks to the inherent clinical and molecular heterogeneity of cancer we now know that exists and to our inability to predict the behavior of any particular tumor. And so the question can be legitimately posed: will TNM survive the molecular revolution [33]? We feel that is unlikely to change for the foreseeable future. In large part, the place of TNM remains secure if only for the fact that newer biological findings will need to be evaluated in the context of an existing robust structure such as that provided by TNM, even if it remains imperfect. In addition, TNM is also a worldwide language, at least in head and neck cancer, and it is not possible to replace it in many areas of the world where complex molecular assays are unavailable. It also represents the basis for entry and stratification in many clinical trials [7] to permit the evaluation of new treatments and biomarkers in a manner that reduces the influence of treatment-selection bias. In head and neck cancer, as in all other regions, we are confronted by a large group of potential factors, but their precise place in the management of the disease remains uncertain. Articles are appearing that address a bewildering multitude of potential molecular characterizations of head and neck cancers, often in studies containing only modest patient numbers [34–37]. It is not the purpose of this chapter to discuss these in detail but broad comments may be useful as we continue to search for the best use of potential biomarkers

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and explore how to incorporate these important elements that have the potential to profile these tumors in methods that take us beyond pure extent of disease. In the paragraphs that follow, for squamous cell carcinoma of mucosal origin, we have chosen two relatively well-recognized biomarkers, specifically the expression of the epidermal growth factor receptor (EGFR) and of HPV, that could be readily available if needed for clinical management of patients with head and neck cancer in the developed world. Both have been discussed by the head and neck task forces in the process of preparation of the seventh edition TNM. The situations surrounding both biomarkers will be discussed in relation to the proposition that they could replace or enhance the TNM or other prognostic models in the near future. For some time, it has been recognized that EGFR expression is an independent determinant of survival and a robust independent predictor of loco-regional relapse, although not for distant metastasis that is capable of withstanding the scrutiny of rigorous multivariate analysis. However, in one of the original landmark correlative studies of a large series of patients treated with radiotherapy alone, EGFR expression varied considerably among head and neck squamous cell carcinomas, and the study was restricted to the investigation of higher stage patients (i.e., in excess of 95% of patients had UICC/AJCC Stage III or IV disease) [38]. Thus, the precise impact of this biomarker in the continuum of the different degrees of head and neck cancer disease extension remains unclear. This problem in fact exists in much of the prognostic factor literature, where different factors or prognostic models may be important in subsets of a disease that address issues such as advanced stage as compared to early disease, or in different scenarios (e.g., primary vs. recurrent presentations) but it becomes problematic when one wishes to apply them universally across the entire disease spectrum. An additional problem relating to EGFR expression concerns its true value in the clinic as matters stand today. The initial data suggested that EGFR expression might be considered for selecting patients for more aggressive combined therapies or enrollment into trials targeting EGFR signaling pathways [38]. Strong claims have persisted that it is a promising therapeutic target in head and neck cancer based on the proven efficacy of cetuximab, a monoclonal antibody against EGFR, when combined with radiotherapy in locally advanced cancer (Stages III and IV) [39]. This observation had led to the approval of the drug for this indication on a worldwide basis. However, the role of EGFR-targeting agents in other therapeutic modalities, such as combined chemoradiotherapy or induction chemotherapy, remains to be defined [40]. In addition, and perhaps more disheartening, is the knowledge that the useful effects of cetuximab appear to be divorced from the degree of EGFR expression [40, 41]. The reality is that the majority of squamous cell carcinomas in the head and neck overexpress EGFR, but the clinical responses to

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EGFR-targeting agents have been modest, and molecular predictors for response to EGFR-targeted therapies have not been identified in the head and neck. Molecular marker studies have shown that mutations in the EGFR gene such as the L858R mutation in the tyrosine kinase portion of the receptor confer sensitivity to EGFR tyrosine kinase inhibitors in non-small cell lung cancer, but positive similar and additional studies in head and neck cancer have proven elusive to this point [40]. One opportunity is that emerging data suggest that cetuximab may have the ability to elicit immune responses such as antibody-dependent cell toxicity (ADCC), and the search for predictive biomarkers for cetuximab therapy may need to be redefined to include elements of the immune system. Certainly, the response to cetuximab appears to be multifaceted and involves more than a simple inhibition of the EGFR pathway [40], and until the situation becomes clearer and its role more certain the incorporation of this potentially important biomarker with elements of the TNM remains unresolved. It does seem clear, however, that its place in the prediction of prognosis in head and neck cancer should continue to be evaluated within the established framework of anatomic disease extent and failure to do so may lead to spurious findings. In contrast, the AJCC has recently recommended that HPV status in tumor should be assessed in mucosal squamous cell carcinoma of head and neck sites because of the impact it has on the prognosis of some head and neck cancers [1]. These data, together with other factors not included in TNM, will be compiled in the CS for analysis in the future and in particular as it relates to prognostic models that take into account various factors. This is an encouraging opportunity since the HPV status could influence treatment decisions in the near future due to modifications in treatment intensity currently under consideration [42]. However, even HPV status needs to be viewed cautiously in the present discussion. In essence, the landscape of head and neck cancer has been dramatically altered in recent years by the emergence of this subgroup of cancers with a different oncogenesis compared to the traditional situation where tobacco and alcohol were almost the sole etiologic agents. Strong epidemiologic evidence exists for the rapidly increasing incidence of a specific subgroup of squamous cell carcinomas, predominantly in the tonsil and base of tongue regions of the oropharynx, that are associated with HPV oncogenesis [43]. These tumors seem to have significantly more favorable outcome compared to traditional squamous cell cancer in these locations [44]. This has been attributed to enhanced immunological response to HPV in the host or to an increased sensitivity to radiotherapy [42] though evidence suggests that outcome is also related to better response following both chemotherapy and surgery. These findings have led to HPV being widely accepted as a prognostic biomarker for oropharyngeal carcinomas.

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An alternative interpretation is to regard this as an entirely different disease compared to traditional oropharynx cancer. In essence, it remains unresolved whether it should be considered separately from traditional smoking-related oropharyngeal cancer, and clinically trials are now being designed specifically with this in mind to tailor treatment strategies to these more favorable cancers. This potentially implies that a different TNM classification could even be considered in this disease and would be akin to the way a disease such as NPC is approached where its different etiology, also predominantly viral, and case profile set it aside from other head and neck cancer. Apart from their different etiology, other evidence for considering HPV-related oropharyngeal cancers uniquely includes the characteristic histological description of these tumors as poorly differentiated, often exhibiting minimal keratinization, basaloid features, and exhibiting clinical features that include noninvasive submucosal primary lesions, lymph nodes with palpable features that resemble those found in lymphoma patients, and that appear cystic on computerized tomography (CT) [45]. Recently, it has even been suggested that lymph node involvement carries dramatically less prognostic importance compared to traditional head and neck cancers emphasizing again that it is difficult to evaluate the influence of these important biomarkers unless the evaluation is undertaken within some framework that addresses the extent of disease. Indeed the evidence appears to be that, in this group of patients, a substantial percentage of whom have metastasis to cervical lymph nodes in less advanced primary tumors, the N status, is an unreliable prognostic indicator [46]. Again this is reminiscent of the NPC situation where different consideration to N-classification has been needed, although the direction of the effect was the opposite due to higher risk of distant metastases in NPC with advanced neck disease. Additional complexity also exists in relation to racial differences in outcomes for oropharyngeal cancer and that is related to molecular basis of these tumors. Recent data suggests that the adverse outcome of black patients compared to white patients may be explained by the paucity of association with HPV expression in tumors among the black population [47]. The precise reason for the disparity in HPV expression remains unresolved but its absence appears strongly associated with significantly less favorable outcome of oropharyngeal cancer in blacks compared to patients where HPV is associated. Finally, in considering the HPV situation, patients who have HPV-related oropharyngeal cancers but who are smokers appear to retain some of the adverse profile of more traditional head and neck cancer and do not fare as well as never smoked patients [36, 48]. Such “hybrid etiology” cancers appear to be complex, and in this situation the concept of a biomarker within the spectrum of regular and traditional oropharyngeal cancer may indeed apply. Complicated interplays exist, including additional adverse expression of EGFR

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that appears to be expressed, possibly through increased hypoxia in the tumor tissues in smokers’ cancers [36]. In addition, in their modest cohort of 66 patients, Kumar et al. identified other unexplained variables including an adverse effect of female gender (although only 12 were female) and additional adverse biomarkers. The authors advised additional validation to understand the role of these findings in predicting and guiding therapies. This would also apply to how these findings could be incorporated with TNM staging. Again most of the patients had presented with relatively advanced regional node involvement or with fairly advanced T-category disease rendering it difficult to address the whole spectrum of the disease [36].

Serum Markers Among mucosal head and neck cancers, NPC has additional uniqueness in possessing a robust circulating tumor marker that can be expected to be employed clinically. One of the uses is the correlation of circulating EBV DNA with disease staging using quantitative real-time polymerase chain reaction (PCR) technology [49]. By means of its production by NPC cells, EBV DNA level has been shown to be more powerful than existing staging system in predicting outcomes by providing an index of disease burden in the individual patient and has been investigated now by numerous authors [50]. In particular, Leung et  al. showed that pretherapy circulating EBV DNA load is an independent prognostic factor for overall survival in NPC. Thus, patients with early stage disease can be segregated by EBV DNA levels into a poor-risk subgroup with survival similar to that of Stage III disease and a good-risk subgroup with survival similar to Stage I disease [51]. While this provides an attractive concept, it also faces challenges in whether it can be applied universally at this time, especially in regions where the disease is most prevalent and resources to make it universally available are not as plentiful as in the developed world. A possibility may be to use it presently as an additional tool within clinical trials to augment prognostic assessment and disease monitoring. Also importantly, while it is attractive to consider it as a molecular marker that provides characterization of disease for prognostication, it falls somewhat short of this. As is the case for prostate specific antigen in prostate cancer staging, or in the case of serum markers for testis cancer, both of which are incorporated in the TNM classification [1, 2], these blood assays are considered indicators of disease burden and, in reality, represent surrogates for disease bulk. The same probably applies in NPC since the influence of the circulating marker correlates with the full spectrum of disease extent and the disparity noted above from Leung et al. could be explained by imprecision in estimating the extent of disease in these complex tumors in the region of the skull base.

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True biological serum markers that represent qualitative tumor characteristics are less common where expression is relevant to the oncogenic process (as opposed to the burden of disease) and is a complex area of research [30]. Some examples in the head and neck have proved unreliable, such as those available in thyroid cancer. Here the situation is aggravated by other dimensions of prognosis that need to be considered. Thus, in differentiated thyroid cancer, discrepancies between methods for the assessment of thyroglobulin may be problematic [52]. Also, more than 95% of papillary and follicular carcinomas are thyroglobulin positive, even those that are metastatic, and well-differentiated tumors generally express more thyroglobulin than poorly differentiated tumors [53]. This therefore means most patients with aggressive tumors and adverse prognosis may not necessarily manifest higher levels of thyroglobulin. On the other hand, in medullary thyroid cancer, biomarkers such as basal calcitonin; stimulated calcitonin, and carcinoembryonic antigen are powerful indicators of tumor activity. However, unlike EBV DNA levels in NPC, the levels of serum calcitonin may not necessarily reflect tumor burden (volume) accurately. Thus, in the view of the authors, they are not perfect [54]. Also, elevated but stable calcitonin levels seem not to portend unfavorable outcome after treatment and a more representative assessment may relate to calcitonin doubling times. These assessments are not available as baseline variables and while potentially prognostic in follow-up do not aid in addressing the needs of baseline assessment and staging [55]. Familial medullary carcinoma was one of the first tumor, where genetic predisposition to the development of cancer was confirmed, by the assessment of RET Protooncogene, in gene carriers. This powerful genetic marker accurately identifies gene carriers, and has facilitated the prevention of ­medullary carcinoma by early elective thyroidectomy during infancy and childhood.

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using a TNM system. Not only is the measurement of tumor volume a tedious process requiring the tumor to be outlined digitally on cross-sectional imaging, but also the results are prone to difficulties created by both intra- and interobserver discrepancy. To overcome this problem, several investigators have developed semiautomated systems to reduce interoperator as well as intraoperator variability [58]. In order to overcome the technical and manpower considerations, alternative simpler methods have also been suggested including standard bidimensional measurements [59, 60]. While there seems to be no doubt that tumor volume provides a robust predictor of outcome in many head and neck cancers, including claims of superiority to TNM in the contemporary era of head and neck cancer treatment, problems with implementing this approach remain. Manpower issues and other problems have not yet been resolved, including the determination of agreed potential cutpoints that might be used to create a classification that meets the needs of the clinician and scientists. This is also particularly relevant in regions of the world where NPC is most prevalent. In the end it must also be acknowledged that while volume assessment could provide utility if it was introduced, it remains fundamentally a measure of the extent of disease. In addition, the tumor volume of a totally exophytic cauliflower-like cancer does not have the same prognostic implications, as a tumor of the same volume, which is nearly all endophytic. It has been a long-standing observation that exophytic tumors are quite radiosensitive, in contrast to endophytic tumors. Thus, tumor volume, such as assessment of serum markers, is not strictly divorced from the anatomic stage paradigm and does not address many of the problems discussed earlier and that seem to lie at the heart of many of the criticisms of TNM [33].

Evolution of Biology with Advancing Stage Volume as a Predictor Classification based on tumor volume instead of strict anatomic extent alone has been reported as a significant prognostic factor in the management of head and neck cancer. In turn, this has prompted investigators to suggest the incorporation of tumor volume into the TNM staging system. Indeed an extensive literature has now emerged that addresses this topic, but will not be discussed exhaustively. Much of this knowledge emanates from the treatment of NPC but has also been reported for other head and neck cancers [56, 57]. Nonetheless, if tumor volume is to be used as an independent prognostic factor, the methods for volume measurement need to be standardized [58]. Unfortunately, the technical challenges to implement this in the clinical setting routinely need to be resolved if it is to be used to classify patients

Another complex problem involving interplay between anatomic disease extent and molecular characterization of disease concerns the potential that disease could evolve in its character as it progresses from early to more advanced stage. While undesirable for patients, and implying the need for more intensive treatment as disease evolves, investigators might readily embrace this concept. Thus, intensified treatment, while often used for anatomically more extensive tumors, could additionally be needed because the disease character has evolved to a more aggressive phenotype. In turn, this also could open the door to the potential for a true molecular-based “staging system.” Unfortunately, while the proposal is attractive in concept, few useful examples are available in the head and neck region. Investigation into this important area will need robust translational science activities, grounded in the laboratory and the clinic, where

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the anatomic stage classification and clinical parameters provide the framework for this evaluation. An example, in laryngeal cancer, is a study intended to address shortcomings in cancer prognostication and treatment due to a lack of methods to adequately address the complexity and diversity of disease. The authors of this study used multiparametric methods to identify specific patterns of disease progression. They investigated, on an exploratory basis, whether genomewide alterations of loss and gain, using a panel of 122 gene probes (112 unique genes), discriminated between early stage (Stages I and II) and late stage (Stages III and IV) laryngeal squamous cell carcinomas. Significant differences between early vs. advanced stage were apparent for the following genes: ERBB4, CASP2, RECQL4, and BCL7A. Loss of ERBB4 (P = 0.045) and BCL7A (P = 0.019) significantly discriminated between early and advanced stages. Gain of RECQL4 copy number (P = 0.043) was associated with advanced stage; gain of CASP2 (P = 0.043) characterized early disease, but loss was associated with advanced stage. Problems with this approach include not only the isolated nature of this study, but also the multiple significance testing makes it important to validate the findings independently. The potential that the number of statistical assessments used could result in spuriously significant observations by chance alone appears to have also been recognized by the authors who identified their study as “exploratory” [61]. A related issue with a different application exists within the domain of head and neck cancer staging that embodies the concept of tumor evolution over time. In essence, this, as in the previous example, relies on the fact that carcinogenesis is a multistep process at both the phenotypic and genetic levels. A malignant neoplasm has several phenotypic attributes which commences with the benign and acquires genetic events that carry it through sequential steps that ultimately lead to excessive growth, local invasion, and the ability to form regional or distant metastases [62]. An application of this evolution with some practical clinical consequence relates to the potential to temporally model some of the key genetic events of a cancer and to identify whether different areas of cancer in the same patient could be related to each other or could have descended from each other. A very practical use for this is the potential to identify if pulmonary squamous cell carcinoma in a patient with head and neck squamous cell carcinoma might represent metastatic disease or a second primary. Depending on the approach taken for these two scenarios, it may have profound implications for a patient who may be denied potentially curative treatment when this might be possible if such a lesion is incorrectly declared metastasis. For some time, the ability has been available to achieve this diagnostic distinction using molecular tools for an important element of cancer staging, but as yet it seems not to have been translated actively to the clinic [63, 64].

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The Importance of Host Factors It has been well recognized that features of the host have significant prognostic impact in head and neck cancer. However, with the exception of differentiated thyroid cancer, where patient age is an important factor, the head and neck TNM classification does not take into account any host characteristics. A consistent feature of the management of laryngeal cancer has been the demonstration that female gender is a powerful and independent favorable factor in addition to other more traditional factors. In a large retrospective series (n = 1,252) from Aarhus University Hospital in Denmark, women had absolute improvements of approximately 10% compared to men for all cancer-specific outcomes including local control, loco-regional, disease-specific survival, and overall survival following curative radiotherapy [65]. Female gender seems to retain this favorable advantage in other sites as well, based on a very large recent series (n = 3,821) from Germany [66]. For this reason, reports of adverse outcome in HPV-related oropharyngeal cancer in women compared to men is unexpected [36, 67]. While these represent small studies, they raise the possibility of host interactions with the biological process underlying the pathogenesis of head and neck cancer and the subsequent response to treatment. Earlier we have also noted the discrepancy in outcome between black vs. white patients with oropharyngeal squamous cell carcinoma and the fact that there is a dramatic difference in the association of cancers in these two groups with HPV oncogenesis, and the precise reasons underlying this remain speculative [47]. There is also evidence that the status of the host immune system may be relevant and may be an explanation for the unusually favorable outcome of HPV-related oropharyngeal cancer compared to traditional cancers in this location [68]. Another well-described host-related prognostic variable for outcome in head and neck cancer is comorbidity. Comorbidity is described as “the presence of one or more medical ailments, in addition to the primary tumor but not caused by the primary tumor” [69]. Risk factors for the development of head and neck squamous cell carcinoma, such as smoking and alcohol abuse, contribute to other diseases as well (e.g., cardiovascular, pulmonary, or hepatic diseases). Therefore, comorbidity is to be expected in these patient groups. This has been well established by early work from Picciliro [70] to more recent reporting of the influence of comorbidity for the first time in hypopharyngeal cancer [71]. Several established validated instruments designed to code and quantify comorbidity are available. These include, in historic order, the cumulative illness rating scale (CIRS) [72], the Kaplan Feinstein comorbidity index (KFI) [73], the Charlson comorbidity index (CCI) [74], and the index of coexistent disease (ICED) [75].

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In a comparative study of these four instruments, the KFI was the most successful in stratifying patients with head and neck cancer [76] though the CIRS appeared to be uniquely robust in another report that addressed laryngeal cancer exclusively managed with surgery [77]. Whether this would apply to patients treated with organ preservation strategies is unclear and emphasizes the context-based nature of some of these analyses that are sometimes overlooked. Nevertheless, a very consistent finding throughout such literature of head and neck cancer is the observation that comorbidity, assessed in various ways, seems to have as significant effect as the stage in understanding the prognosis of patients with these cancers and needs to be considered in designing treatment approaches. These analyses may also provide a framework for amalgamation of the various elements of prognosis into usable prognostic models that may be applicable in a broader perspective. This is discussed below in the section on “Combining Variables and Validation.”

The Importance of Environmental Factors The relationship between outcome and the environment where the patient with head and neck cancer is treated can be profound and the reasons underpinning these can be complex. What sets these apart from other prognostic factors is that they exert influence external to the parameters of the host and tumor but their value relates to their ability to explain reasons for differential outcomes for treatments that might otherwise be expected to be similar. A classification is available and includes factors related to the physician, the health care system, and society [31]. Each can also be subdivided into treatmentrelated issues (e.g., expertise, access, and health care delivery processes), educational issues (e.g., participation in continuing education, development of practice guidelines, and access to information), or quality issues (e.g., quality of treatment, quality of the health care facility, and access to affordable health insurance). Interested readers should consult the original description for a more detailed discussion [31]. The problem of environment as a prognostic factor is well exemplified by the recent report of outcome in a large prospective randomized trial where the technical planning and radiotherapy parameters of almost 700 patients were evaluated by a team of expert head and neck radiation oncologists. This review was undertaken without knowledge of the outcome of the patient or of the arm of the trial on which the patient was treated. In patients who received at least 60  Gy, those with major deficiencies in their treatment plans had a markedly inferior outcome compared with those whose treatment was initially protocol compliant. The 2-year overall survival was 50% vs. 70% (hazard ratio 1.99; P < 0.001) and the 2  years freedom from loco-regional failure was 54% vs. 78% (hazard

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ratio 2.37; P < 0.001) for deficient vs. compliant radiotherapy, respectively. A large variation in the percent of plans with major adverse impact was noted according to country. Even more striking was the correlation between the number of patients entered and the probability of receiving unsatisfactory radiotherapy. In centers enrolling fewer than five patients, 29.8% had a predicted major adverse impact compared with 5.4% in centers enrolling more than 20 patients [78]. Another recent interesting example relates to the availability of modern radiotherapy facilities in the form of access to intensity-modulated radiotherapy (IMRT). The use of IMRT has rapidly become widespread for the delivery of radiotherapy for patients with head and neck cancer in the USA. However, significant geographic variations are apparent in the utilization of IMRT, and patients in census tracks comprising the lowest socioeconomic quartile were less likely to receive IMRT than their more affluent counterparts [79]. Other recent reports also point out disappointing examples of environmental health care disparities associated with advanced head and neck presentations in the USA. These are much more likely to be evident in patients without adequate health care insurance, or individuals, especially blacks, residing in regions with low educational accomplishments or with low median household incomes. Similar findings were seen in patients with laryngeal cancer [80] and oropharyngeal cancer [81]. The authors indicate that it is important to consider the impact of insurance coverage on disease stage at diagnosis and associated morbidity, mortality, and quality of life. Similar findings on stratified analysis and logistic regression were applied to two million incident cancers (1997– 2000) from 32 states representing 57% of the US population. For a great many cancers, poverty as a factor independently predicts advanced stage cancer suggesting improved access and utilization of good medical care might facilitate earlier diagnosis and longer survival [82]. Consistent with these findings is the report of a large series (n = 1,231) of patients with primary squamous cell carcinoma of the oral cavity, pharynx, or larynx diagnosed or treated at the University of Pittsburgh by Kwok et al. [83] They report that patients with Medicaid/uninsured and Medicare disability were at increased risk of death after a diagnosis of SCCHN when compared with patients with private insurance, after adjustment for age, gender, race, smoking, alcohol use, site, socioeconomic status, treatment, and cancer stage. Similarly, Molina and colleagues studied 20,915 patients with head and neck cancer in the Florida Cancer Data System and showed that African American and poor patients have a dramatically worse prognosis although the disparity is not entirely explained by demographics, comorbidity, or undertreatment [84]. While numerous other factors are also associated with adverse outcome, space does not permit a more detailed discussion of this very important and often overlooked area.

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Ironically, as implied by the examples shown above, these factors have the greatest potential for remediation with consequent improvement in outcome compared to other prognostic factors but this can only be accomplished if resource inadequacies and process deficiencies are addressed.

Combining Variables and Validation The science of prognostic factor assessment is a nascent area that needs to be considered in a broader context. We have seen that the dimensions of prognosis in head and neck cancer cover a wide field, yet there remains uncertainty about how to proceed in our goals of using the extent of this knowledge to its full capability. It does appear that critical dismissal of one dimension as being less useful than another is probably not the solution, nor is it helpful to dismantle a system that is being used successfully worldwide, for over 40 years, to permit newer elements to be introduced if the framework was not designed to receive them. In general terms, some agreement on taxonomy and methodology is required. Perhaps the adoption of formal terms such as Staging to describe the anatomic extent of disease and Profiling to describe the qualitative characteristic of tumors may be a start. The use of the term Prognostic models could then ­permit them to be combined in a rational way that allows their full impact to be exploited. These concepts are under active discussion by the UICC and AJCC. Different aspect of these will be discussed below under different rubrics that address the traditional TNM groupings, the use of prognostic indexes, the use of nomograms, and the area of validation and comparison of prognostic models.

Handling Prognostic Groups Within TNM In addressing the need to combine different prognostic elements into groups, the UICC and the AJCC have taken slightly different approaches in the seventh edition TNM classification. The AJCC has substituted the term “Anatomic Stage/Prognostic Groups” in place of what were previously termed “Stage Groups” when the elements of TNM are combined together within the TNM in the seventh edition [1]. However, the goal of the new terminology is the same as it was previously, i.e., to create a basic form of prognostic index. The UICC has approached this slightly differently in the seventh edition although the intent is identical to the AJCC, namely, to permit the incorporation of validated nonanatomical prognostic factors at present or in the future. The UICC’s approach is to use two forms of grouping of component elements [2]. The predominant one for this edition is termed “Stage Groups” and contains only anatomic factors

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for virtually all sites within TNM and represents the same “Stage Groups” as were used in the former sixth edition. Certain diseases that traditionally used some nonanatomic factors, e.g., thyroid cancer where age has been incorporated, and sarcomas that included grade, are retained in the “Stage Groups” of the seventh edition to avoid disruption to a classification developed many years ago. However, the incorporation of newer nonanatomic factors is being addressed by the creation of a third dimension within the UICC’s version of TNM in the form of “Prognostic Groups.” In truth, these are identical to the AJCC’s “Anatomic Stage/Prognostic Groups” in the few diseases where this applies and for all other diseases the UICC “Stage Groups” are analogous. At present only two diseases have the new “Prognostic Groups” in the UICC version, namely, prostate and esophageal cancer, in both of which pathological grade was recently introduced in the classification. There are currently no head and neck sites included in this process. In time, it is possible that the UICC may also modify thyroid and sarcoma so that the anatomic and nonanatomic elements will only be aggregated together in the “Prognostic Groups,” and the “Stage Groups” will only contain anatomic extent of disease variables throughout TNM. In this way, anatomic disease extent can be addressed independently in “Stage Groups” or in combination with nonanatomic factors in the “Prognostic Groups,” the latter being analogous to the “Anatomic Stage/Prognostic Groups” of the AJCC. A final and more sobering dimension in the area of “Prognostic Groups” or “Stage Groups” is the fact that these are generally developed in a pragmatic rather than pure ­scientific way. Hence, the literature contains numerous examples of the theme that the TNM stage group classifications, while successful in creating statistically distinct groups, often do not perform as well as other stage grouping systems [85]. Potentially, the future will require some attention to this area of research as well if the groups formulated within the classification are to be considered seriously. Detailed discussion of alternative staging systems is reported in the literature [86].

Prognostic Indexes The head and neck literature contains a growing body of reports devoted to combining different elements of prognosis together. Generally, the intention is to focus on a particular setting (e.g., previously untreated patients, patients with recurrent cancer, patients with metastatic disease, early stage disease vs. more advanced disease, etc.). Usually, the intention is to facilitate decision-making in the management of patients, usually concerning some intervention that is typically treatment related. Behind most is the goal of generating

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a quantified prognosis in the form of a score that may be of use to the patient, guiding clinical decisions, and may be useful for guiding eligibility for clinical trials tailored to specific treatments and patient types. Some of the dimensions are appropriate to combine together, but some may be less so in the sense that they may contain elements that are not present at baseline time of diagnosis and treatment decision-making even though they may be characterized as such. A typical example is the inclusion of the status of resection margins in a model where this variable only becomes available after the first and often most important treatment has been administered (namely, surgery). Thus, it is not only unavailable at baseline, but it also automatically selects out cases with different prognosis based on their likelihood of undergoing a successful resection with clear margins. Cases with positive resection margins can be expected to be already having adverse prognosis from the stand-point of the anatomic extent of disease, but such classifications may still be highly useful in guiding decision-making for the use of adjuvant treatments once the primary treatment has been undertaken. This further illustrates the theme that disease extent must be considered in applying prognostic models, and one cannot necessarily extrapolate to another setting whether it concerns different stages of disease, different anatomic sites, or different scenarios (e.g., primary vs. recurrent cancer). There is insufficient opportunity to explore the different models that have been developed in the head and neck arena but these include, among others, attention to parotid cancer [87, 88], metastatic nasopharyngeal cancer [89], laryngeal cancer [77], hypopharyngeal cancer [71], and various combinations of cancers of the larynx, oral cavity, and pharynx [69]. Some of these studies were mentioned earlier in the context of comorbidity in the section on “The Importance of Host Factors” where many have included comorbidity assessed in various ways combined with the TNM and other elements of anatomic disease extent and included other factors such as age, gender, and some pathological features. As yet there is no report that incorporates a robust model that combines molecular characterization of disease (or even host) with more traditional domains and this type of work is very inviting for the future. As noted some studies have combined different prognostic factors that include biological markers with more traditional parameters such as gender and smoking but they have not as yet been formulated into a prognostic index to guide decision-making for individual patients or even groups of patients [36, 48].

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into a single numerical estimate of the probability of an event that is tailored to the profile of an individual patient. Often these use appealing graphical interfaces, commonly displayed by computer, that facilitate interaction with individual patients about their personal disease situation. While widely used in some areas of oncology, especially prostate cancer, there is very little published material concerning nomograms for head and neck cancer. Gross et  al. developed a nomogram for guiding adjuvant treatment after surgery for oral cavity squamous cell carcinoma [90]. Notably, this was developed for relatively early stage resected oral cancer and this context must be remembered as it is easy to stray from the original basis of the nomogram when using it to discuss problems with patients. So far there is no evidence that this is happening in head and neck cancer but there may be such instances in other diseases. The AJCC, in particular, is exploring the use of nomograms to address the potential goal of creating a “continuous prognostic nomogram” for each site and each patient, where the anatomic TNM staging will remain as the fundamental factor, but other important features, such as biomarkers as well as comorbidities, will be included with a weighted score to arrive at a “prognostic score,” at any given point throughout the patient’s life [10]. In this concept, the prognostic score will be a dynamic “staging and prognostic” tool to accurately reflect each patient’s prognosis at the point of inquiry. The CS approach implemented by the AJCC will act as a repository of all available prognostic information for current and future use to support this approach. This ambitious project is potentially both welcome and problematic. Clearly, it is important to be able to encompass the multiple dimensions of prognosis in this way and the concept is certainly meritorious. On the other hand, a limitation is that it largely relates to individual prognosis at this time and additional development will be needed to address groups of patients since one of the goals of the stage classifications is to be able to compare results across groups, in trials, and among regions. Another challenge concerns the statistical underpinnings of these models that require careful scrutiny, including the degree of uncertainty surrounding the point estimates. This is thoroughly addressed in a recent review that includes cautionary language that the methodology underlying the construction of nomograms should be understood by clinical users so that prognostic estimates are appropriately communicated [91].

Validation and Comparison of Prognostic Models Nomograms Nomograms are widely used for cancer prognosis, primarily because of their ability to reduce statistical predictive models

An important aspect of the creation of prognostic indexes concerns the underlying statistical principles and the epidemiological basis for their creation. This area cannot be

8  Head and Neck Cancer Staging and Prognosis

addressed here but the reader should be aware of such principles as the generalizability of the index to patients outside the source population. It includes transportability of results beyond the domain where it was created such as transportability regarding geographic location, but also by time or era, which may be more difficult to address with different historical dimension to the data, its assembly, and its use. Other dimensions include clinical and statistical validation. The complex nature of these issues and the assumptions behind the models, including understanding their inherent weaknesses, require attention and are summarized more completely elsewhere [87, 88, 92, 93]. Other elements in understanding prognostic models, and especially when comparing models against each other, concern a variety of concepts in the evaluating process. These include hazard consistency (i.e., homogeneity within strata for the outcome of interest), hazard discrimination (i.e., each stratum chosen should have a statistically distinct prognosis compared to the stratum above and below it for the outcome), outcome prediction (i.e., maximizing prediction accuracy by techniques such as percent of variation in outcome explained by the scheme or by measuring the slope, or degree of separation in the mean probability predictions), and balance (where different prognostic strata or groups are relatively even and balanced). These are detailed elsewhere for the interested reader [86, 94].

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B. O’Sullivan and J.P. Shah 54. Machens A, Dralle H. Biomarker-based risk stratification for previously untreated medullary thyroid cancer. J Clin Endocrinol Metab. 2010;95(6):2655–63. 55. Boostrom SY, Grant CS, Thompson GB, et al. Need for a revised staging consensus in medullary thyroid carcinoma. Arch Surg. 2009;144(7):663–9. 56. Chong VF. Tumour volume measurement in head and neck cancer. Cancer Imaging. 2007;7 Spec No A:S47–49. 57. Studer G, Lutolf UM, El-Bassiouni M, Rousson V, Glanzmann C. Volumetric staging (VS) is superior to TNM and AJCC staging in predicting outcome of head and neck cancer treated with IMRT. Acta Oncol. 2007;46(3):386–94. 58. Chong VF, Ong CK. Nasopharyngeal carcinoma. Eur J Radiol. 2008;66(3):437–47. 59. King AD, Zee B, Yuen EH, et al. Nasopharyngeal cancers: which method should be used to measure these irregularly shaped tumors on cross-sectional imaging? Int J Radiat Oncol Biol Phys. 2007;69(1):148–54. 60. Lee CC, Ho HC, Su YC, et  al. Bidimensional measurement of nasopharyngeal carcinoma: a simple method to predict outcomes. Clin Otolaryngol. 2009;34(1):26–33. 61. Saglam O, Shah V, Worsham MJ. Molecular differentiation of early and late stage laryngeal squamous cell carcinoma: an exploratory analysis. Diagn Mol Pathol. 2007;16(4):218–21. 62. Jones S, Chen WD, Parmigiani G, et  al. Comparative lesion sequencing provides insights into tumor evolution. Proc Natl Acad Sci USA. 2008;105(11):4283–8. 63. Geurts TW, Nederlof PM, van den Brekel MW, et  al. Pulmonary squamous cell carcinoma following head and neck squamous cell carcinoma: metastasis or second primary? Clin Cancer Res. 2005;11(18):6608–14. 64. Leong PP, Rezai B, Koch WM, et al. Distinguishing second primary tumors from lung metastases in patients with head and neck squamous cell carcinoma. J Natl Cancer Inst. 1998;90(13):972–7. 65. Johansen LV, Grau C, Overgaard J. Laryngeal carcinoma – multivariate analysis of prognostic factors in 1252 consecutive patients treated with primary radiotherapy. Acta Oncol. 2003;42(7):771–8. 66. Guntinas-Lichius O, Wendt T, Buentzel J, et al. Head and neck cancer in Germany: a site-specific analysis of survival of the Thuringian cancer registration database. J Cancer Res Clin Oncol. 2009. 67. Hannisdal K, Schjolberg A, De Angelis PM, Boysen M, Clausen OP. Human papillomavirus (HPV)-positive tonsillar carcinomas are frequent and have a favourable prognosis in males in Norway. Acta Otolaryngol. 2010;130(2):293–9. 68. Dahlstrand H, Dahlgren L, Lindquist D, Munck-Wikland E, Dalianis T. Presence of human papillomavirus in tonsillar cancer is a favourable prognostic factor for clinical outcome. Anticancer Res. 2004;24(3b):1829–35. 69. Datema FR, Ferrier MB, van der Schroeff MP, Baatenburg de Jong RJ. Impact of comorbidity on short-term mortality and overall survival of head and neck cancer patients. Head Neck. 2010;32(6):728–36. 70. Piccirillo JF, Feinstein AR. Clinical symptoms and comorbidity: significance for the prognostic classification of cancer. Cancer. 1996;77(5):834–42. 71. Homma A, Sakashita T, Oridate N, et al. Importance of comorbidity in hypopharyngeal cancer. Head Neck. 2010;32(2):148–53. 72. Linn BS, Linn MW, Gurel L. Cumulative illness rating scale. J Am Geriatr Soc. 1968;16(5):622–6. 73. Kaplan MH, Feinstein AR. The importance of classifying initial co-morbidity in evaluation the outcome of diabetes mellitus. J Chronic Dis. 1974;27(7–8):387–404. 74. Charlson ME, Pompei P, Ales KL, MacKenzie CR. A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis. 1987;40(5):373–83. 75. Cleary PD, Greenfield S, Mulley AG, et al. Variations in length of stay and outcomes for six medical and surgical conditions in Massachusetts and California. JAMA. 1991;266(1):73–9.

8  Head and Neck Cancer Staging and Prognosis 76. Hall SF, Rochon PA, Streiner DL, Paszat LF, Groome PA, Rohland SL. Measuring comorbidity in patients with head and neck cancer. Laryngoscope. 2002;112(11):1988–96. 77. Castro MA, Dedivitis RA, Ribeiro KC. Comorbidity measurement in patients with laryngeal squamous cell carcinoma. ORL J Otorhinolaryngol Relat Spec. 2007;69(3):146–52. 78. Peters LJ, O’Sullivan B, Giralt J, et  al. Critical impact of radiotherapy protocol compliance and quality in the treatment of advanced head and neck cancer: results from TROG 02.02. J Clin Oncol. 2010;28(18):2996–3001. 79. Guadagnolo BA, Liu C-C, Cormier JN, Du XL. Evaluation of trends in the use of intensity-modulated radiotherapy for head and neck cancer from 2000 through 2005: socioeconomic disparity and geographic variation in a large population-based cohort. Cancer. 2010;116(14):3505–12. 80. Chen AY, Schrag NM, Halpern M, Stewart A, Ward EM. Health insurance and stage at diagnosis of laryngeal cancer: does insurance type predict stage at diagnosis? Arch Otolaryngol Head Neck Surg. 2007;133(8):784–90. 81. Chen AY, Schrag NM, Halpern MT, Ward EM. The impact of health insurance status on stage at diagnosis of oropharyngeal cancer. Cancer. 2007;110(2):395–402. 82. Greenlee RT, Howe HL. County-level poverty and distant stage cancer in the United States. Cancer Causes Control. 2009; 20(6):989–1000. 83. Kwok J, Langevin SM, Argiris A, Grandis JR, Gooding WE, Taioli E. The impact of health insurance status on the survival of patients with head and neck cancer. Cancer. 2009;116(2):476–85. 84. Molina MA, Cheung MC, Perez EA, et al. African American and poor patients have a dramatically worse prognosis for head and neck cancer: an examination of 20,915 patients. Cancer. 2008; 113(10):2797–806.

155 85. Hall SF, Groome PA, Irish J, O’Sullivan B. TNM-based stage groupings in head and neck cancer: application in cancer of the hypopharynx. Head Neck. 2009;31(1):1–8. 86. Patel SG, Lydiatt WM. Staging of head and neck cancers: is it time to change the balance between the ideal and the practical? J Surg Oncol. 2008;97(8):653–7. 87. Poorten VV, Hart A, Vauterin T, et al. Prognostic index for patients with parotid carcinoma: international external validation in a Belgian-German database. Cancer. 2009;115(3):540–50. 88. Vander Poorten VL, Hart AA, van der Laan BF, et al. Prognostic index for patients with parotid carcinoma: external validation using the nationwide 1985–1994 Dutch Head and Neck Oncology Cooperative Group database. Cancer. 2003;97(6): 1453–63. 89. Toh CK, Heng D, Ong YK, Leong SS, Wee J, Tan EH. Validation of a new prognostic index score for disseminated nasopharyngeal carcinoma. Br J Cancer. 2005;92(8):1382–7. 90. Gross ND, Patel SG, Carvalho AL, et al. Nomogram for deciding adjuvant treatment after surgery for oral cavity squamous cell carcinoma. Head Neck. 2008;30(10):1352–60. 91. Iasonos A, Schrag D, Raj GV, Panageas KS. How to build and interpret a nomogram for cancer prognosis. J Clin Oncol. 2008; 26(8):1364–70. 92. Harrell Jr FE, Lee KL, Mark DB. Multivariable prognostic models: issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors. Stat Med. 1996;15(4):361–87. 93. Justice AC, Covinsky KE, Berlin JA. Assessing the generalizability of prognostic information. Ann Intern Med. 1999;130(6):515–24. 94. Groome PA, Schulze KM, Mackillop WJ, et al. A comparison of published head and neck stage groupings in carcinomas of the tonsillar region. Cancer. 2001;92(6):1484–94.

Chapter 9

Biomarkers in Head and Neck Cancer John Wrangle, Shanthi Marur, and Arlene A. Forastiere

Abstract  Biomarker research provides the opportunity to risk stratify patients based on identified prognostic and predictive markers. The need for such biomarkers is ­evident to improve response and survival outcomes in head and neck cancer through more rational patient selection for intensive curative regimens as well as palliative treatments. Increasing numbers of molecularly targeted therapeutics are in preclinical and clinical evaluation in head and neck cancer. These compounds, added to current chemotherapy and radiotherapy regimens, are costly and potentially add to normal tissue toxicity. This chapter focuses on markers with potential for testing in large validation clinical trials. As yet, no one marker has validated predictive capacity of utility in the selection of therapy for individuals with head and neck cancer. HPV-16 appears to be prognostic for better outcome while high EGFR expression is prognostic for poor outcome. Validated diagnostic tests that are widely available and collaboration among investigators are additional future challenges in biomarker research for head and neck cancer. Keywords  Molecular biomarkers • Head and neck cancer • Therapeutic targets • Predictive and prognostic markers

Introduction The management of patients with head and neck cancer has changed dramatically over the past 30 years from a ­surgically dominated specialty to a multidisciplinary decision-making approach. Nearly all patients presenting with locally advanced cancers now receive chemotherapy combined with radiotherapy as a part of their treatment, often as a strategy to preserve organ function or as an adjuvant following surgery. Advances have also occurred in radiation technology for treatment

A.A. Forastiere (*) Oncology Department, Johns Hopkins University, School of Medicine, Bunting/Blaustein Cancer Research Building, 1650 Orleans Street, Room G90, Baltimore, MD 21231, USA e-mail: [email protected]

p­ lanning and dose delivery to improve local ­control and reduce the volume of normal tissue treated and risk of late effects. The entrance of molecularly targeted therapeutics into the clinical arena offers an exciting opportunity to improve upon the outcomes achievable with standard ­cytotoxic ­regimens and radiotherapy. However, these ­therapies have high cost and may add to the existing toxicity risk profile of chemoradiation. The current system of histopathologic and radiographic assessments of tumor stage and surgical margins has limited ability to stratify patients for specific risk of metastasis, ­local–regional recurrence or the development of a second ­primary. The identification of molecular predictive markers is a logical and rational next step to achieve more than a ­marginal improvement in outcome within the coming decade. This would allow tailoring of treatment based on risk – treatment associated with high morbidity would ideally be recommended for patients at the highest risk of dying from their cancer, thus limiting exposure. High cost novel biologic therapies would be utilized based on individual biomarker tumor characteristics. The underpinnings already exist for biomarker-driven research as great strides have been made in the understanding of ­carcinogenesis in head and neck cancer. Progression from early carcinogenesis, including premalignant lesions to ­invasive squamous cell carcinoma is known to be a multistep process of genetic alterations and has been studied extensively in tobacco-related head and neck squamous cell cancers (HNSCC) [1]. Alterations in cyclin D1, p53, and p16 tumor suppressor genes are early events of carcinogenesis. Alterations in signaling pathways, growth factors, angiogenesis, cell ­adhesion, and cell death characterize later events of carcinogenesis leading to invasion and metastasis. Biomarker research is a critical next step in discovery. The ideal biomarker would have three characteristics. First, it would have the ability to accurately diagnose cancer and recurrence in asymptomatic populations. Second, the biomarker would be prognostic, that is, to have the ability to predict the risk of recurrence or survival or be predictive of response to a specific therapy. Third, the biomarker would also serve as a therapeutic target. Research has focused on molecular markers of cell-cycle control (cyclin D-1, Rb gene product, p16, p21, p27),

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Table 9.1  Predictive markers Predictive marker

Predicts

Study (year)

N

HR for Response Response Lab OR progression HR for to CT to CT-RT PFS OS DFS method (p-value) (CI) death (CI) (p-value) (p-value) (p-value) (p-value) (p-value)

EGFR EGFR

Cetuximab sensitivity Burtness (2005) [8] 117 IHC Cetuximab sensitivity Vermorken 442 IHC (2008) [9]

0.05 –

EGFR p16 p53 p53 Bcl-xL Bcl-xL Bcl-xL Bcl-2 ERCC-1 ERCC-1

Response to therapy Response to therapy Response to therapy Larynx preservation Larynx preservation Complete response Larynx preservation Local control Cisplatin sensitivity Cisplatin sensitivity

HPV

Response to initial chemotherapy HPV Response to chemoradiotherapy RASSF1A/2A Response methylation to radiation TIMP3 Response methylation to radiation CDH1 Response methylation to radiation

IHC IHC IHC IHC IHC IHC IHC IHC IHC IHC

– – – – – – –

– 0.75 (0.59– 0.95) – – – – – – –

– –

– –

– –

0.055 0.009 – – – – –

– – – – – – –

– –

0.01 0.008 0.002 0.03 0.02 0.143 0.06 >0.0016 – –

– 0.01

– –

– –

0.036 –

Kumar (2008) [44] Kumar (2008) [44] Temam (2000) [67] Kumar (2008) [44] Kumar (2008) [44] Trask (2002) [60] Trask (2002) [60] Wilson (1996) [59] Jun (2008) [75] Handra-Luca (2007) [70] Worden (2006) [76]

42 PCR

–

–

–

0.001

–

–

Worden (2006) [76]

42 PCR

–

–

–

0.005

–

–

Huang (2009) [73]

482 MSP

De Schutter (2009) [77] De Schutter (2009) [77]

70 70 99 70 70 47 47 93 45 96

– 0.47 (0.37– 0.61) – – – – – – –

0.009

59 MSP

0.005

0.12

43 MSP

0.002

0.007

Table 9.2  Prognostic markers Study (year)

Prognostic marker

Kumar (2008) [44] Kumar (2008) [61] Akervall (1997) [51] Marsit (2008) [54]

↓HPV titer ↓p53/↑Bcl-xL 11q13 rearrangement CCND1

Akervall (1997) [51] Marsit (2008) [54] Michalides (1997) [53] Namazie (2002) [55] Ang (2002) [78] Chung (2006) [14] Kumar (2008) [44] Rubin Grandis (1998) [79] Temam (2007) [13] Xia (1999) [80] Xia (1999) [80] Kumar (2008) [44] Kumar (2008) [44] Poeta (2007) [66]

Cyclin D1 Cyclin D1 Cyclin D1 Cyclin D1 amplification/p16 deletion EGFR EGFR EGFR EGFR EGFR EGFR/HER-2/HER-3 HER-2 p16 p53 p53

Rubin Grandis (1998) [79] Pena (1999) [58] Wilson (1996) [59] Kumar (2008) [44] Gillison (2000) [33] Licitra (2006) [81] Worden (2008) [76] Marsit (2008) [74]

TGFa Bcl-2 Bcl-2 HPV HPV HPV HPV CDH-1 methylation

91 42 93 50 253 90 42 340

N

75 444

Lab method

75 109 115 103

Cytogenetics Sequence detection IHC IHC IHC FISH

155 86

IHC FISH

91 134 47 47 50 50 420

IHC RT-PCR IHC IHC IHC DNA microarray IHC IHC IHC PCR PCR PCR PCR MSP

DFS

OS

PFS

LP

HR for survival (CI)

– – – –

0.008 0.005 0.005 –

– – – –

– – – –

– – – 0.6 (0.4–1.0)

– – –

0.047 – 0.05 0.003

– – –

– – –

0.0016 – – 0.0001 0.0001 – – – – –

0.0006 <0.01 0.001 0.0001 0.0001 <0.05 <0.001 0.001 – –

– <0.05 – – – – – – – –

– – – – – – – – 0.03 –

1.7 (1.2–2.4)

0.0001 <0.05 – – – – –

0.0001 – 0.012 0.03

– – – – – – –

– – – – – – –

– – – – 0.41 (0.20–0.88) – –

0.0018 0.007 <0.02

– 3.6 (1.0–13.0) – – – – – – – – –

9  Biomarkers in Head and Neck Cancer

apoptosis (p53, Bcl-xl, Bcl-2), growth regulation (HER family – EGFR and downstream pathways, Cox-2, Ki-67), and measures of hypoxia (CA9, HIF-1a). Potentially useful molecular markers in head and neck cancer also include ­detection of viral DNA – the human papillomavirus (HPV), predominantly in oropharynx cancer and Epstein-Barr virus (EBV) associated with nasopharyngeal carcinoma. This ­chapter focuses on biomarkers specific to HNSCC, where there is a body of literature indicating clinical utility or high potential for clinical utility as ­prognostic or predictive markers (Tables 9.1 and 9.2).

159

The Epidermal Growth Factor Receptor (EGFR) is a ­transmembrane glycoprotein and a member of the ErbB family of receptors. This receptor is composed of an extracellular ligand-binding domain, a transmembrane region and an intracellular domain that includes the tyrosine kinase enzyme. Endogenous ligands, particularly transforming growth factoralpha (TGFa), stimulate the external receptor to undergo conformational change and dimerization [2]. Dimerization results in activation of intracellular tyrosine kinase, protein phosphorylation and stimulation of various cell signaling pathways that mediate cell cycle progression, angiogenesis, inhibition of apoptosis, tumor invasion, and metastasis (Fig. 9.1). EGFR is a prognostic marker for disease-free and overall survival of HNSCC. The majority of HNSCC (~90‰) overexpress EGFR relative to normal tissue; EGFR expression

level is associated with poor prognosis and decreased overall survival [3, 4]. Overexpression of EGFR’s primary ligand, TGFa is associated with poor response to anti-EGFR therapy and overall poor prognosis [4, 5]. The most commonly used method of EGFR protein detection is immunohistochemistry (IHC), which is a practical diagnostic tool that can be performed in most laboratories. However, the limitations of IHC include inter- and intra-variability in scoring by pathologists and lack of standardization of IHC assays. A more informative method uses automated quantitative analysis (AQUA) of immunofluorescence. EGFR expression can be scored on membrane, cytosolic, and nuclear components. Using the AQUA immunofluorescence technique, investigators showed that both tumor EGFR and nuclear EGFR staining were independent prognostic factors [6]. EGFR is also a molecular target for therapeutic intervention in HNSCC as well as colorectal cancer, and nonsmall cell lung cancer. Two classes of drugs target the EGFR, antibodies bind to the extracellular ligand binding domain while small molecule tyrosine kinase inhibitors (TKI) bind to the intracellular tyrosine kinase domain. Cetuximab, a chimeric monoclonal IgG1 antibody directed against EGFR, is FDAapproved in combination with radiotherapy for locally advanced HNSCC [7]. Cetuximab, when combined with ­cisplatin, demonstrated modest activity in previously treated recurrent or metastatic head and neck cancer [8] and survival benefit when used as first line therapy in combination with platinum-based chemotherapy in metastatic or recurrent head and neck cancer [9]. As single agents, antibodies directed against EGFR, and small molecule TKI of EGFR have

Fig.  9.1  EGFR and the mode of action of cetuximab. Binding of a ligand to EGFR causes receptor dimerization, either with another EGFR monomer or with another member of the erbB family, leading to tyrosine kinase activation. The resultant receptor autophosphorylation initiates signal-transduction cascades involved in cell proliferation and

survival. Cetuximab blocks binding of ligands to EGFR, inhibiting receptor phosphorylation and downstream events. Modified from Kirkpatrick P, Graham J, Muhsin M. Cetuximab. Nature Reviews Drug Discovery 2004:549–550. Adapted by permission from Macmillian Publishers Ltd. © 2004

Epidermal Growth Factor Receptor

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­ odest activity as measured by standard clinical response m criteria (partial response rate 4–10%) [10, 11]. EGFR overexpression has not been shown to correlate with the efficacy of EGFR-targeted therapy in HNSCC and therefore does not serve as a predictive biomarker [11, 12]. The mechanisms that believed responsible for the low response to EGFR inhibition provide insight into potential prognostic and predictive biomarkers and therapeutic targets. These include signaling pathways that are independent of EGFR, such as EGFR nuclear localization and direct ­transactivation of genes, ERK and AKT activation by the insulin growth factor receptor-1 (IGFR-1), Src/STATmediated transactivation of EGFR; gene mutations causing dysregulated cell cycle check points, such as EGFR variant III (EGFR vIII) mutation in the extracellular domain, and EGFR mutation of the kinase domain. EGFR gene amplification is under evaluation as a ­prognostic factor in HNSCC. The EGFR gene is amplified in 10–58% of HNSCC and is measured by fluorescence in situ hybridization (FISH) and quantitative PCR. In two independent studies, EGFR gene amplification was associated with worse progression-free and overall survival [13, 14]. EGFR gene amplification is positively associated with response to EGFR-directed antibody therapies in nonsmall cell lung ­cancer and colon cancer [15] but as yet, there are no ­published

reports correlating gene amplification with response outcome to EGFR-targeted therapies in HNSCC. Similarly, EGFR polymorphisms (on EGFR intron 1) in the extracellular domain have been found in nonsmall cell lung cancer and colon cancer [16, 17] and are associated with significantly improved PFS and OS in colon cancer [18]. This is another potential prognostic biomarker of interest in HNSCC. The most common mutation of the extracellular ligand binding domain is the inframe deletion of exons 2–7, known as EGFR variant III and is reported in about 42% of HNSCC. This mutation is detected by RT-PCR [19]. In preclinical studies, the EGFR vIII mutation was associated with poor response to anti-EGFR agents, but this is not yet a proven predictive marker in the clinical setting in HNSCC. Upregulated EGFR-dependent pathways drive tumor proliferation and survival. Two main pathways are the RAS/ RAF/MAP kinase signaling pathway for proliferation and the PI3K/AKT signaling pathway for cell survival. Activation of these pathways independent of EGFR is known to be a major cause of resistance to anti-EGFR agents. Three ­pathways that have been the focus of investigations as potential therapeutic targets, prognostic and predictive biomarkers are the PI3K-AKT/mTOR pathway, Src/STAT pathway, and IGFR pathway (Fig. 9.2) [20]. The protein products of these signaling intermediates can be measured to determine gene

Fig.  9.2  Possible resistance to epidermal growth factor receptor (EGFR) inhibition in head and neck squamous cell carcinoma (HNSCC) as a result of EGFR-independent activation of oncogenes. In addition to oncogenic pathways activated by EGFR, EGFR-independent oncogenesis in HNSCC may result from activation of G protein-couples receptors (GPCRs), other growth factor receptors, genetic ­aberrations, such as mutation and amplification of oncogenes, loss of tumor suppressor genes, and vascular endothelial growth factor

(VEGF) overexpression promoting angiogenesis. MMP matrix metalloproteinase, IGF-1R insulin-like growth factor-1receptor, VEGFR VEGF receptor, PKC protein kinase, STAT signal transducer and activator of transcription, Rb retinoblastoma. From Kalyankrishna S, Grandis JR. Epidermal Growth Factor Receptor Biology in Head and Neck Cancer. Journal of Clinical Oncology 24:2666–2672, 2006. Reprinted with permission © 2006 American Society of Clinical Oncology. All rights reserved

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activation or repression in response to anti-EGFR ­therapies and provide a rationale for the use of multitargeted biologic therapies.

PI3 Kinase/AKT Pathway and mTOR Signaling through the PI3K/AKT pathway leads to ­activation of the mammalian target of rapamycin (mTOR). mTOR is represented by two structurally and functionally distinct ­multiprotein signaling complexes, mTORC1 (mTOR ­complex 1, rapamycin sensitive) and mTORC2 [21]. mTORC1 is mainly activated via the PI3 kinase pathway through AKT. mTORC2 is activated through a currently unknown mechanism, possibly by receptor tyrosine kinase (RTK) signaling. It has been suggested that mTORC2 phosphorylates and activates a different pool of AKT that is not upstream of mTORC1. mTORC2 is rapamycin insensitive. PI3K is inhibited by PTEN, and acts as a tumor suppressor by regulating the AKT pathway [22]. The PI3K/AKT/mTOR pathway is dysregulated by loss/mutation of the PTEN through PI3K mutation/ amplification; through AKT/PKB overexpression/overactivation; mutation of PIK3CA is associated with reduced response rate following anti-EGFR therapeutics in metastatic colon cancer. These mutations are noted in a very small population approximately 8% in HNSCC [23]. Loss of PTEN expression either by hypermethylation or loss of heterzygosity (LOH) of PTEN occurs in 12% of HNSCC and could contribute to resistance seen with anti-EGFR agents [24]. Hence, mutation of PIK3CA and loss of PTEN and markers of PIK3/AKT pathway are b­ iomarkers with p­ redictive potential.

IGFR/AKT Pathway The type I insulin-like growth factor receptor (IGF-IR) is a member of a family of transmembrane tyrosine kinases and is activated by two high affinity binding ligands, insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) [25]. Unlike many cell surface receptors, the IGF-IR exists as a preformed dimer; it requires domain rearrangements rather than receptor oligomerization for cell signaling. Upon ligand binding, the IGF-IR undergoes a conformational change, thereby activating its tyrosine kinase activity. The principal pathways for transduction of the IGF signal are the mitogen-activated protein kinase (MAPK) and ­phosphatidylinositol 3-kinase (PI3K)/AKT pathways [26]. There appears to be crosstalk between EGFR and IGF-1R and resistance to anti-EGFR agents is secondary to persistent IGF-1R mediated PIK3/AKT activation [27, 28]. Preclinical studies of IGF-IR TKI OSI868, alone and in combination

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with an EGFR TKI (erlotinib), showed low response in cell lines with higher levels of pIGF-IR or EGFR expression exposed to single one inhibitor and significantly enhanced anti-proliferative effect to the combined EGFR TKI and IGF-1R TK [29]. Based on these observations, clinical trials combining biologic therapeutics to target both EGFR and IGFR are in progress.

Src/STAT Pathway The Src-STAT pathway is activated in response to EGFR activation [30]. Activated Src-kinases release RTK ligands that activate the Raf-ERK/MAPK pathway and represent another mechanism to circumvent EGFR inhibition. These pathways as with EGFR, influence cell adhesion, migration, angiogenesis, differentiation, proliferation, and cell apoptosis. Dysregulation and overexpression of Src leads to tumorigenesis, tumor progression, and metastasis. Increased phospho-Src (p-Src), and decreased E-cadherin in HNSCC was associated with more invasive aggressive tumors, poor ­differentiation, and more frequent lymph node metastases [31]. Dasatinib, a dual Src/Abl kinase inhibitor has shown inhibition of migration and invasion in HNSCC cell lines [32]. Clinical trials combining Src-targeted agents and EGFRtargeted agents for the treatment of metastatic HNSCC and combined with radiotherapy for an initial curative treatment are in progress. Correlative studies evaluating receptor ­activation status, downregulation by the targeted therapeutic and correlation with outcome variables provides insight into the potential utility of Src as a prognostic and predictive biomarker.

HPV HPV is commonly associated with anogenital cancers and more recently has been associated with a subset of HNSCC [33]. There are over 100 types of HPV virus, but only about 15 are considered to have oncogenic potential. The HPV type that is most frequently associated with HNSCC is t HPV-16; it is also associated with cervical and vulvar ­cancers in women; anal and penile cancers in men and women [34]. Over the past decade, the incidence of oropharynx cancers has shown an upward trend in young men in the USA and Europe in the absence of exposure to two major risk factors, tobacco and alcohol [35]. HPV-16 is now recognized to be a prognostic factor for survival outcome. HPVrelated cancers appear to show increased sensitivity to radiotherapy and ­chemotherapy, but HPV-16 has not been validated as a ­predictor of response to specific therapies.

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HPV is a sexually transmitted virus. The major risk factors appear to be an increased number of lifetime oral sex and vaginal sex partners and possibly marijuana use. HPV-associated HNSCC has a distinct risk factor profile, clinical presentation, biology, and tumor morphology [36, 37]. The incidence of HPV-associated head and neck cancer varies geographically, worldwide. The highest rates are observed in Sweden with approximately 90% of oropharynx cancers associated with HPV, approximately 60% in the USA, 30–40% in Western Europe and very low, unchanging rates in Southeast Asia. Patients with HPV positive head and neck cancer ­commonly present with large cystic neck nodes and a small ­primary (T1) in the tonsil or base of the tongue. Histologically, these cancers are usually nonkeratinized, poorly differentiated squamous cancers with basaloid features [38, 39]. HPV-16 detected by FISH or p16 detected by IHC have been shown in retrospective and prospective studies to be prognostic for a favorable outcome relative to patients with HPV negative oropharynx cancer. When treated with chemotherapy and radiation therapy, these HPV positive patients appear to be more responsive to these treatments translating to a significantly better overall survival and progression free survival outcome [40]. The biology of HPV-associated HNSCC is distinct from the tobacco-associated HNSCC and could explain the favorable outcomes observed with HPV-associated HNSCC [41]. The HPV virus integrates its DNA into the host cell nucleus, encodes for E6 and E7 genes and dysregulates the production of E6 and E7 oncoproteins. The E6 oncoprotein ­promotes

Fig. 9.3  HPV infection and cell cycle dysregulation. HPV DNA ­integrates into the host genome and produces oncoproteins E6 and E7. E6 inhibits wild-type p53 function through ubiquitination, releasing cell-cycle arrest. E7 upregulates p16 and downregulates Cyclin D1 via inactivation of retinoblastoma tumor suppressor to promote proliferation and malignant ­transformation. From Gillison M. Human papillomavirus-associated head and neck cancer is a distinct epidemiologic, clinical, and molecular entity. Sem Oncol 2004;31(6):11. Reprinted with kind permission from Elsevier

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ubiquitanation and degradation of p53 promoting cell ­survival. The E7 oncoprotein binds and inactivates the retinoblastoma tumor suppressor gene leading to upregulation of p16 and low expression of cyclin D1 cell cycle disruption, proliferation, and malignant transformation (Fig. 9.3). The method preferred for HPV detection is FISH, instead of PCR which requires a sophisticated technology and fresh frozen tissue [42]. The prognostic impact of HPV-associated head and neck cancer has defined a distinct subset of oropharynx cancer and future clinical trials are stratified for this ­factor or there are separate trials designed for HPV positive and HPV negative oropharynx cancers. Retrospective studies have explored the association between HPV status, p16, tobacco exposure and survival [43]. The ­survival outcomes for HPV-16 positive and p16 positive patients were similar. The pattern of failure revealed significantly lower rates of locoregional failure, in HPV ­positive HNSCC patients compared to HPV negative patients and no difference in the distant metastases between the two groups. When survival was assessed after adjusting for tobacco exposure the HPV positive <20 pack year smokers had a significantly better 2  year overall survival (95%) ­compared to HPV negative ³ 20 pack year smokers (63%) suggesting that tobacco exposure altered the biology of HPV positive tumors. Other investigators have reported a positive correlation between the combination of low EGFR expression, high p16 expression and improved survival outcome in contrast to patients who had high EGFR expression and low HPV titer or high EGFR and low p16 expression [44].

9  Biomarkers in Head and Neck Cancer

NF-kB The transcription factor Nuclear Factor-kB (NF-kB) is a ­protein complex that acts on promoter sites involved in many cellular processes, including survival and apoptosis, inflammation, migration, cell proliferation, and angiogenesis. It is present ubiquitously in human cells and serves as a point of convergence for several signaling pathways. As a rapid acting transcription factor, NF-kB is normally converted from its inactive form to an active transcription factor in response to a cellular insult. The inactive state of NF-kB is maintained by Inhibitor of Kappa Beta (ikB) and requires the activity of a proteasome for activation. In HNSCC, tobacco is a major risk factor, and there is evidence suggesting that cigarette smoke condensate activates NF-kB via direct phosphorylation and subsequent degradation of IkB. The list of inciting stimuli which lead to activation and subsequent homo- and heterodimerization of NF-kB and its homologs are diverse and include bacterial lipopolysaccharide, viruses, and fungi as well as cytotoxic agents, DNA damage, and radiation. The activation of NF-kB results in activation of receptors and signaling proteins, including IL-1/ interleukin-1 receptor 1 (IL-1R1) and TGFa/EGFR in HNSCC [45]. Alterations in the PI3-K/Akt pathway, such as mutations of PI3-K or loss of PTEN, cause persistent activation of NFКB and ongoing oncogenesis. Hypoxia and ionizing radiation induce NF-kB activity [46, 47]. Inhibition of activation by combining targeted therapy such proteosome inhibitors and inhibitors of upstream targets that may contribute to aberrant NF-kB activation or coactivated pathways may have therapeutic potential for HNSCC. In HNSCC cells, treatment with bortezomib down modulates expression of NF-kB-regulated genes cyclin D1, Bcl-XL and IAP-1, inhibits cellular proliferation and angiogenesis, and promotes apoptosis [48, 49]. Biomarkers of NFkB activation, biomarkers of inhibition of NF-kB, and inflammatory markers are under investigation in clinical trials that combine proteosome inhibitors with other targeted therapeutics, chemotherapy, and radiation therapy. In squamous cell cancer of the larynx, NF-kB status as determined by IHC positively correlated with nodal status and T stage, and low expression was associated with poor outcomes [50]. The ultimate utility of NF-kB may be in its predictive capacities. It may be useful not only for selecting therapies specifically targeting increased NF-kB activation, but also predicting response to cytotoxic agents and radiation.

Cyclin D1 Studies of Cyclin D1 show that this regulator of cell cycle transition from G1 to S phase may have prognostic implications in HNSCC. Polymorphisms in CCND1, the gene

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encoding Cyclin D1, have been associated with many aspects of the natural history of the disease, from the risk of developing HNSCC, grade of the tumor, risk for recurrence in operable disease, and overall survival [51–54]. Cyclin D1 acts after pairing with cyclin-dependant kinases 4 and 6 (CDK4, CDK6). This interaction may be further perturbed by inactivation of p16, an inhibitor of CDK4 and 6. Inactivation or deletion of p16 has been associated with poor prognosis and, conversely, relatively high levels of p16 are associated with greater response to therapy [44, 54, 55]. The activation of cyclin D and related complexes is important for cells to enter into S phase and for the G1–S transition of the cell cycle. The Cyclin D Kinase (CDKN2A) locus, located on chromosome 9p21, encodes two functionally distinct tumor suppressor genes, p14ARF and p16INK4a, which play an active role in the p53 and Rb tumor suppressor pathways. Any alteration of p16 disrupts the expression of cyclin D1 causing abnormal proliferation of the cells. In tobacco-related HNSCC, p16 loss is well documented and cyclin D1 is overexpressed and/or amplified and has been correlated with more advanced, aggressive disease, lymph node metastasis, and reduced survival [55, 56]. By contrast, patients with HPV-associated HNSCC, characterized by overexpression of p16 and low levels of cyclin D1, exhibit high response rates to chemotherapy and chemoradiation, and apparent longer progression-free and overall survival [57].

Bcl-2 Bcl-2 and Bcl-xL are members of the Bcl-2 family of 25 genes. Bcl-2 was the first gene described as having a role in apoptosis, or programed cell death. Originally studied in B-cell lymphomas, hence its name, the Bcl-2 gene is placed next to the immunoglobulin heavy chain locus in a ­characteristic chromosomal translocation, [14, 18]. The ­juxtaposition results in dramatic overexpression of the Bcl-2 gene product resulting in dysregulation of the normal proand anti-apoptotic balance. Bcl-2 overexpression is noted in a variety of tumors, where this translocation does not occur and there are likely multiple other mechanisms involved. The way in which Bcl-2, Bcl-xL, and other members of this family exert their influence over apoptosis is not clear, though they may act by regulating the release of apoptotic factor by forming ion channels in the outer membrane of mitochondria. Programed cell death occurs in response to a variety of insults, such as starvation, irreparable DNA damage, or viral infection and is intended to lead to an orderly elimination of a damaged cell in a way not hazardous to the organism it belongs to. A variety of soluble factors influence whether cells undergo apoptosis, some promoting and others inhibiting it. Bcl-2 and Bcl-xL are both considered

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a­ nti-apoptotic agents, and about 70% of all HNSCC upregulate one of the two [58]. Paradoxically, Bcl-2 overexpression has been associated with improved outcomes in a HNSCC tumor’s response to therapy, local control, time to ­progression, and survival underscoring the lack of certainty [58–60]. Reduced expression of Bcl-xL was associated with better outcomes in larynx preservation [60, 61]. Like many other cellular pathways with no directed drug target, the bcl-2 family of genes has predictive value for therapies not known to act on them specifically.

p53 and DNA Damage Repair Discovered in 1979 and characterized as a tumor suppressor in 1983, p53 is a highly studied, critical element of cell cycle regulation and is mutated in over half of all human malignancies [62]. The normal role of p53 is to respond to an ­enormous variety of stress signals, classically hypoxia and DNA ­damage [62, 63]. Bound to HDM2 p53 is inactive, but cleavage in response to these various stress signals may be induced, as well as decreased degradation. As a result, the quantity of p53 cell increases. Transcription of TP53, the gene encoding p53, is upregulated in response to stress signals from the MAPK family of protein kinases and many other kinases involved specifically in checkpoint integrity. Once activated, p53 initiates cell cycle arrest at the G1/S transition to allow for DNA repair [62, 63]. If DNA repair occurs, the cell is allowed to reenter the cell cycle. If this pause in cell division fails to restore the integrity of the DNA, cell death ensues. Mutations of TP53 occur at high frequency in human ­cancer in general, and HNSCC in particular. Estimates of the prevalence of this mutation in HNSCC range from about 40% to 80%, and if the prevalence of aberrant p53 ­expression is estimated when including the nonmutational, ­downregulating effects of HPV E6 protein, the estimate approaches 100% [64]. Note, though, that p53 aberrations, whether by mutation or variation in expression level, may not be prognostically equivalent. Complicating this further, all mutations may not be equivalent, where certain nucleotide substitutions in certain exons of TP53 lead to a spectrum of inactivation from no effect on activity to total functional inactivation. For instance, a patient with a proline for arginine substitution at codon 72 of exon 4 may do worse than those with wild-type TP53 [65]. Mutational status in aggregate, however, has been studied both as a prognostic and predictive marker, and tumors with mutated p53 appear less likely to respond to neoadjuvant chemotherapy and associate with decreased overall survival [66, 67]. More recently, Perrone et  al. looked retrospectively at banked tumor samples and showed that 40% of patients with wild-type p53 or mutations which left partial function had

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complete response to chemotherapy with cisplatin/fluorouricil (no taxanes used) versus 10% in those with nonfunctional mutants [68]. The bewildering variety of potential prognostic and predictive significance of even a single gene is highlighted by p53 and highlights the need for well-designed validation studies and likelihood of complex interpretation of an individual’s data, especially when paired with the many other markers that are obtained in tandem. The role of p53 is that of cell cycle regulator, and as such is responsible for initiating important DNA damage repair mechanisms. An enzyme essential to repairing cross-linked DNA adducts created by akylating agents, such as cisplatin is the excision repair cross-complementation group 1 (ERCC1). Having become the subject of intense interest since studies showed that its overexpression is associated with lack of response to cisplatin-containing regimens in nonsmall cell lung cancer, it has since been shown to be a potential marker for response to therapy and overall survival in HNSCC [69, 70].

Methylation Epigenetic modifications are increasingly understood and of increasing prognostic and predictive relevance. Normally functioning DNA methyltransferases silence genes by methylating promoter regions. Thought to be an early step in ­carcinogenesis, abnormal methylation activity results in inactivation of tumor suppressor genes contributing to carcinogenesis, metastasis, invasiveness, and deregulation of the cell cycle. Methylation status has prognostic and therapeutic potential. The concept of demethylating therapy started in myleodysplastic syndrome and is being adapted to solid tumors trials. Many tumors have well-characterized, high incidence of hypermethylation at specific promoter regions. Hypermethylation in HNSCCs has been demonstrated at many genes, including those of p16, E-cadherin, MGMT, DAPK1, RAR beta, and cyclin A1 among many [71, 72]. The Ras/P13K/AKT pathway is thought to be a major factor in radiation sensitivity, and ­hypermethylation of RASSF1A and RASSF2A in a study of 482 samples was correlated with response to radiation [73]. Methylation of the CDH-1 promoter may have prognostic ­significance having been observed to correlate with overall ­survival [74].

Conclusion Current research and patient care are influenced by rapidly advancing knowledge of the molecular biology of head and neck cancer, and the complexity of interconnecting pathways

9  Biomarkers in Head and Neck Cancer

from cell surface receptors to transcriptional activation of genes mediating uncontrolled cellular proliferation and ­survival. Molecular target identification and an array of new therapeutics present challenges to the standard methodologies for clinical trial design, evaluation of efficacy and toxicity. Risk stratification based on molecular prognostic and predictive markers is next on the horizon for advancing the field. This chapter has focused on markers with potential for testing in large validation clinical trials. As yet, no one marker has validated predictive capacity of utility in the selection of therapy for individuals with head and neck ­cancer. HPV-16 appears to be prognostic for better outcome while high EGFR expression is prognostic for poor outcome. Validated diagnostic tests that are widely available and ­collaboration among investigators are additional future challenges in biomarker research for head and neck cancer.

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J. Wrangle et al. 52. Holley SL, Matthias C, Jahnke V, et al. Association of cyclin D1 polymorphism with increased susceptibility to oral squamous cell carcinoma. Oral Oncol. 2005;41:156–60. 53. Michalides RJ, van Veelen NM, Kristel PM, et al. Overexpression of cyclin D1 indicates a poor prognosis in squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg. 1997;123:497–502. 54. Marsit CJ, Black CC, Posner MR, et  al. A genotype-phenotype examination of cyclin D1 on risk and outcome of squamous cell carcinoma of the head and neck. Clin Cancer Res. 2008;14: 2371–7. 55. Namazie A, Alavi S, Olopade OI, et al. Cyclin D1 amplification and p16(MTS1/CDK4I) deletion correlate with poor prognosis in head and neck tumors. Laryngoscope. 2002;112:472–81. 56. Okami K, Reed AL, Cairns P, et al. Cyclin D1 amplification is independent of p16 inactivation in head and neck squamous cell carcinoma. Oncogene. 1999;18:3541–5. 57. Pyeon D, Newton MA, Lambert PF, et al. Fundamental differences in cell cycle deregulation in human papillomavirus-positive and human papillomavirus-negative head/neck and cervical cancers. Cancer Res. 2007;67:4605–19. 58. Pena JC, Thompson CB, Recant W, et al. Bcl-xL and Bcl-2 expression in squamous cell carcinoma of the head and neck. Cancer. 1999;85:164–70. 59. Wilson GD, Grover R, Richman PI, et al. Bcl-2 expression correlates with favourable outcome in head and neck cancer treated by accelerated radiotherapy. Anticancer Res. 1996;16:2403–8. 60. Trask DK, Wolf GT, Bradford CR, et al. Expression of Bcl-2 family proteins in advanced laryngeal squamous cell carcinoma: correlation with response to chemotherapy and organ preservation. Laryngoscope. 2002;112:638–44. 61. Kumar B, Cordell KG, D’Silva N, et  al. Expression of p53 and Bcl-xL as predictive markers for larynx preservation in advanced laryngeal cancer. Arch Otolaryngol Head Neck Surg. 2008;134:363–9. 62. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997;88:323–31. 63. Foulkes WD. p53 – master and commander. N Engl J Med. 2007;357:2539–41. 64. Balz V, Scheckenbach K, Gotte K, et al. Is the p53 inactivation frequency in squamous cell carcinomas of the head and neck underestimated? Analysis of p53 exons 2-11 and human papillomavirus 16/18 E6 transcripts in 123 unselected tumor specimens. Cancer Res. 2003;63:1188–91. 65. Sullivan A, Syed N, Gasco M, et al. Polymorphism in wild-type p53 modulates response to chemotherapy in vitro and in vivo. Oncogene. 2004;23:3328–37. 66. Poeta ML, Manola J, Goldwasser MA, et al. TP53 mutations and survival in squamous-cell carcinoma of the head and neck. N Engl J Med. 2007;357:2552–61. 67. Temam S, Flahault A, Perie S, et al. p53 gene status as a predictor of tumor response to induction chemotherapy of patients with locoregionally advanced squamous cell carcinomas of the head and neck. J Clin Oncol. 2000;18:385–94. 68. Perrone F, Bossi P, Cortelazzi B, et al. TP53 mutations and pathological complete response to neoadjuvant cisplatin/fluorouracil chemotherapy in resected oral cavity squamous cell carcinoma. J Clin Oncol. 2009. 69. Reed E. ERCC1 measurements in clinical oncology. N Engl J Med. 2006;355:1054–5. 70. Handra-Luca A, Hernandez J, Mountzios G, et al. Excision repair cross complementation group 1 immunohistochemical expression predicts objective response and cancer-specific survival in patients treated by Cisplatin-based induction chemotherapy for locally advanced head and neck squamous cell carcinoma. Clin Cancer Res. 2007;13:3855–9.

9  Biomarkers in Head and Neck Cancer 71. Ha PK, Califano JA. Promoter methylation and inactivation of tumour-suppressor genes in oral squamous-cell carcinoma. Lancet Oncol. 2006;7:77–82. 72. Shaw RJ, Liloglou T, Rogers SN, et  al. Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br J Cancer. 2006;94:561–8. 73. Huang KH, Huang SF, Chen IH, et al. Methylation of RASSF1A, RASSF2A, and HIN-1 is associated with poor outcome after radiotherapy, but not surgery, in oral squamous cell carcinoma. Clin Cancer Res. 2009;15:4174–80. 74. Marsit CJ, Posner MR, McClean MD, et al. Hypermethylation of E-cadherin is an independent predictor of improved survival in head and neck squamous cell carcinoma. Cancer. 2008;113:1566–71. 75. Jun HJ, Ahn MJ, Kim HS, et al. ERCC1 expression as a predictive marker of squamous cell carcinoma of the head and neck treated with cisplatin-based concurrent chemoradiation. Br J Cancer. 2008;99:167–72. 76. Worden FP, Kumar B, Lee JS, et al. Chemoselection as a strategy for organ preservation in advanced oropharynx cancer: response

167 and survival positively associated with HPV16 copy number. J Clin Oncol. 2008;26:3138–46. 77. De Schutter H, Geeraerts H, Verbeken E, et al. Promoter methylation of TIMP3 and CDH1 predicts better outcome in head and neck squamous cell carcinoma treated by radiotherapy only. Oncol Rep. 2009;21:507–13. 78. Ang KK, Berkey BA, Tu X, et al. Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma. Cancer Res. 2002;62: 7350–6. 79. Rubin Grandis J, Melhem MF, Gooding WE, et al. Levels of TGFalpha and EGFR protein in head and neck squamous cell carcinoma and patient survival. J Natl Cancer Inst. 1998;90:824–32. 80. Xia W, Lau YK, Zhang HZ, et al. Combination of EGFR, HER-2/ neu, and HER-3 is a stronger predictor for the outcome of oral squamous cell carcinoma than any individual family members. Clin Cancer Res. 1999;5:4164–74. 81. Licitra L, Perrone F, Bossi P, et al. High-risk human papillomavirus affects prognosis in patients with surgically treated oropharyngeal squamous cell carcinoma. J Clin Oncol. 2006;24:5630–6.

Chapter 10

Hypoxia in Head and Neck Cancers: Clinical Relevance and Treatment Yungan Tao and Jean Bourhis

Abstract  Tumor hypoxia, or the condition of low oxygen, is a key factor for tumor progression and treatment resistance. Hypoxic areas arise as a result of an imbalance between the supply and consumption of oxygen. Cellular responses to hypoxia are orchestrated through activation of the hypoxia-inducible factor family of transcription factors (HIFs). There are several approaches for detecting tumor hypoxia in head and neck cancers (HNC). Direct oxygen measurements in tissues with Eppendorf-pO2 histography have been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as HIF-1 and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques. Hypoxia appears to be prognostic for outcome in HNC. Several studies have shown that low pO2 in tumor, high HIF-1, Glut-1 and CA-IX expression, serum level of osteopontin correlated with treatment outcomes in HNC patients treated with RT or chemoradiotherapy. Several strategies have been used to overcome hypoxiainduced treatment resistance in HNC, such as hyperbaric oxygen treatment, accelerated radiotherapy with carbogen and nicotinamide, hypoxic cell radiosensitizers: nitroimidazoles, erythropoietin manipulation, and hypoxic cell cytotoxin. More recently, Micro-Environment-Vascular Normalization, HIF-1 Targeting and 18F-FMISO positron emission tomography-based intensity-modulated radiotherapy are promising methods. Keywords  Hypoxia • Radiotherapy • Head and neck cancer • HIF-1

Tumor hypoxia, or the condition of low oxygen, is a key factor for tumor progression and treatment resistance. Hypoxia develops in solid tumors due to aberrant blood vessel formation, fluctuation in blood flow, and increasing oxygen J. Bourhis (*) Radiotherapy Department, Institut Gustave Roussy, 39 Rue Camille-Desmoulins, Villejuif 94805, France e-mail: [email protected]

demands for tumor growth. Because hypoxic tumor cells are more resistant to ionizing radiation, tumor hypoxia has been recognized as a potential cause of failure when treating human solid tumors with ionizing radiation, both in experimental models and in patients with several type of cancer including head and neck cancers (HNC). The importance of hypoxia as a potential mechanism limiting the probability of cure rate in patients with HNC treated with radiation has been recognized [1].

Description of Factors Associated with Hypoxia and Potential Mechanisms of Resistance Related to Hypoxia Hypoxic areas arise as a result of an imbalance between the supply and consumption of oxygen. In locally advanced solid tumors, the O2 consumption rate of neoplastic cells may outweigh a restricted oxygen supply and results in the development of tissue areas with low or very low O2 levels [2]. Other mechanisms are involved in the development of hypoxia in solid tumors: severe structural and functional abnormalities of tumor microvessels induce perfusion limited O2 delivery; deterioration of diffusion geometry limits oxygen penetration; tumor-associated and/or therapy-induced anemia could lead to a reduced O2 transport capacity [2]. As a consequence of these mechanisms tumor hypoxia is associated with the production of fewer radiation-induced cytotoxic free radicals, less radiation-induced DNA damage, and decreased tumor cell kill. This is called as oxygen enhancement effect. Damage to DNA is mainly induced by interaction with oxygen reacting free radicals formed by the ionization of water surrounding the DNA [3]. Typically, DNA strand breaks that are not repaired can lead to fatal chromosomal aberrations. It has been shown that oxygenated cells are 2.5–3 times more radiosensitive than hypoxic cells [3]. Hypoxic cells are also considered to be resistant to most anticancer drugs for several reasons [4]: first, hypoxic cells are distant from blood vessels and, as a result, may not be

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adequately exposed to some types of anticancer drugs [5]; second, cellular proliferation decreases as a function of distance from blood vessels, an effect that is at least partially due to hypoxia; third, hypoxia can select for cells that have lost sensitivity to p53-mediated apoptosis, which might lessen sensitivity to some anticancer agents; fourth; hypoxia can upregulate genes involved in drug resistance, including genes encoding P-170 glycoprotein. Hypoxia is not only an important cause of treatment resistance but also a powerful stimulus of several critical tumor phenotypes. These discoveries have prompted to question whether the link between hypoxia and radioresistance is completely explainable by the oxygen enhancement effect as described above or, whether hypoxia also influences radiosensitivity through biological effects.

Molecular Pathways Involved in Hypoxia Cellular responses to hypoxia are orchestrated through activation of the hypoxia-inducible factor family of transcription factors (HIFs) [6]. HIF-1 is a heterodimer that consists of the hypoxic response factor HIF-1a and the constitutively expressed HIF-1b [7]. The level of HIF-1a expression is determined by the rates of protein synthesis and protein degradation. HIF-1a protein synthesis is regulated via O2-independent mechanisms, by the activation of the phosphatidylinositol 3-kinase (PI3K) and ERK-mitogen-activated protein kinase (MAPK) pathways [8]. These pathways can be activated by signaling via receptor tyrosine kinases, nonreceptor tyrosine kinases, or G-protein-coupled receptors. HIF-1a protein degradation is regulated by O2-dependent prolyl hydroxylation, which targets the protein for ubiquitylation by E3 ubiquitin-protein ligases. These ligases contain the Von Hippel–Lindau (VHL) tumor-suppressor protein, which binds specifically to hydroxylated HIF-1a. Ubiquitylated HIF-1a is rapidly degraded by the proteasome. In the absence of oxygen, HIF-1 binds to hypoxiaresponse elements (HREs), thereby activating the expression of numerous hypoxia-response genes, such as the proangiogenic growth factor vascular endothelial growth factor (VEGF). The redox active apurinic/apyrimidinic endonuclease-1 has been shown to keep HIF-1a in a reduced state that is necessary for its transcriptional function. HIF-1 can affect several intracellular processes, including glycolysis, cell proliferation, apoptosis, angiogenesis, and invasion/metastasis – which have been shown to influence the response to radiation and might, therefore, serve as a link between HIF-1 activity and tumor radiosensitivity. Recently, two other pathways that independently influence gene expression and processes of importance for tumor

Y. Tao and J. Bourhis

cell behavior have proved to be O2-sensitive [9]. The first occurs through regulation of an important integrator of metabolic signals, the kinase mammalian target of rapamycin (mTOR, also known as FRAP1), and its downstream effectors that orchestrate the initiation of protein synthesis, autophagy, and apoptosis sensitivity. The second is through activation of the unfolded protein response (UPR), a program of transcriptional and translational changes that occurs as a consequence of endoplasmic reticulum (ER) stress. The UPR controls multiple downstream processes, including protein production, protein maturation and degradation, cell metabolism, and cell death. HIF-, mTOR- and UPR-dependent responses to hypoxia act in an integrated way, influencing each other and common downstream pathways that affect gene expression, metabolism, cell survival, tumorigenesis, and tumor growth. Increased HIF-1a protein synthesis was inhibited by treatment with rapamycin – a macrolide antibiotic inhibits mTOR. However, it is not known whether phosphorylation of these proteins by mTOR is necessary or sufficient for increased HIF-1a synthesis. In addition to effects on HIF-1a synthesis, activation of the RAF–MEK–ERK signaling pathway has also been shown to stimulate HIF-1a transactivation-domain function. This effect is due at least in part to phosphorylation by ERK of the co-activator p300. A recently characterized hypoxia-induced protein, that regulated in development and DNA damage 1 (REDD1), could negatively control mTOR activity. In head and neck squamous cell carcinoma (HNSCC) cell lines, the expression of the phosphorylated forms of the mTOR downstream targets S6 kinase and S6 (pS6) decreased after hypoxia. These events were associated with REDD1 upregulation. Inhibition of AMP-activated protein kinase (AMPK) before prolonged hypoxia prevented REDD1 expression, thereby sustaining mTOR activity. Reduced mTOR activity in response to hypoxia through AMPK/REDD1 was deregulated, which hence might contribute to the persistent activation of the mTOR pathway in HNSCC cells [10].

How to Detect Hypoxia in the Tumors: Techniques to Measure Tumor Hypoxia There are several approaches for detecting tumor hypoxia in HNC. In a recent hypoxia workshop, convened by Cancer Imaging Program of the National Cancer Institute (NCI) [11], the conclusion was that, although hypoxia is an important aspect of tumor physiology and response to treatment, there is a lack of simple and efficient methods to measure hypoxia and image oxygenation hampers further understanding and limits their prognostic usefulness. There is no gold standard for measuring hypoxia. Briefly, techniques for

10  Hypoxia in Head and Neck Cancers: Clinical Relevance and Treatment

measuring tumor oxygenation can be categorized into two groups: direct invasive and indirect noninvasive methods. Direct oxygen measurements in tissues with Eppendorf-pO2 histography have been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as HIF-1 and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques. The workshop report also presented a comprehensive review of different approaches for measuring tumor hypoxia. Electrode pO2 measurements have been used in several normal tissues, such as brain, breast, subcutis, and skeletal muscle, and these measurements have been used to develop profiles that can be illustrated by pO2 histograms reflecting the oxygenation status of a given tissue. These pO2 distribution profiles may reflect the efficacy of several oxygen supply determinants, such as blood flow rate, the blood’s oxygen transport capacity, the availability of oxygen to the cells, rate of oxygen extraction from the blood, oxygen diffusion distances, microvascular density, and oxygen diffusion coefficients within the tissue, as well as the rate of oxygen consumption by the cells. Although the microelectrode technique directly measures tumor pO2, it suffers from several drawbacks that make it difficult for general use. These include invasiveness, tumor inaccessibility, pressure dependence, interobserver variability, failure to distinguish necrosis from hypoxia, and the lack of spatial information [12]. Endogenous and secreted molecular markers for tumor hypoxia represent proteins and genes whose expressions are induced by hypoxic exposure. One of the most studied oxygen response pathways is HIF-1 pathway, HIF-1 and several of its downstream targets, including Glut-1 (glucose transporter-1), CA-IX, and VEGF, have been widely investigated as prognostic markers in HNC patients with mixed results. One advantage of endogenous markers is that levels of these proteins can be assessed on archival materials, thereby allowing possible correlation with treatment outcomes. In addition, it requires neither the injection of a hypoxic marker drug used as an exogenous nor any additional invasive procedure except the need of a biopsy at diagnosis. A possible drawback of these approaches is that these proteins can be regulated by factors other than hypoxia. Another hypoxia-related marker, the serum level of osteopontin (OPN) has also been reported recently. Le et al. [13] investigated the relationship between OPN, tumor pO2, and prognosis in patients with HNSCC, and it has been shown that Plasma OPN levels appeared to correlate with tumor hypoxia in HNSCC patients and may serve as noninvasive tests to identify patients at high risk for tumor recurrence. Indirect approaches use injectable molecular reporters of oxygen (exogenous marker), which include 2-nitroimidazole compounds, such as misonidazole (MISO), pimonidazole (1-(2-nitro-1-imidazolyl)-3-N-piperidino-2-propanolol) [14],

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and EF5 (nitroimidazole [2-(2-nitro-1H-imidazol-1-yl)N-(2,2,3,3,3-pentafluoropropyl) acetamide]) [15]. These ­compounds form stable adducts with intracellular macromolecules only in hypoxic regions (pO2 < 10  mmHg) [16]. Detection of these adducts with antibodies can provide ­information on the relative oxygenation at the cellular level [17, 18]. In general, 2-nitroimidazole markers stain for areas  of chronic hypoxia and are more sensitive at severe hypoxic conditions than the microelectrode [19]. This approach is limited by the requirement for exogenous drug administration, additional biopsies for staining, and quantification of staining [20]. MRI can provide a useful way to measure hypoxia. Absolute pO2 can be measured on the basis of fluorocarbon reporter molecules. These may be introduced by direct intratumoral injection and they provide measurements consistent with electrodes (interstitial pO2). A major advantage over electrodes is that maps of regional pO2 may be measured at 50–150 individual locations simultaneously. MRI methods for interrogating tumor oxygenation are attractive since they avoid the complication of short-lived radioactivity and MRI equipment is widely available. Blood oxygen level dependent (BOLD) MRI is an imaging technique that distinguishes paramagnetic deoxy-Hb from O2Hb. BOLD MRI signal is related to vascular oxygenation and may allow direct estimates of pO2. However, the correlation becomes difficult for small blood vessels where partial-volume effects combine vessel and tissue in individual voxels and BOLD may also be confounded by flow effects [21]. Oxygen-sensitive MR reporter molecules have also been developed, generally based on perfluorocarbons (PFCs). Other MRI-based imaging such as, FREDOM (fluorocarbon relaxometry using echoplanar imaging for dynamic oxygen mapping) and PISTOL (proton imaging of silanes for tissue oxygen levels) are also under investigation [21]. Positron emission tomography (PET)-based hypoxia imaging has also been widely over the past 15  years. 18F-fluoro-misonidazole [1-(2-nitroimidazolyl)-2-hydroxy3-fluoropropane; 18F-FMISO], is the most widely used PET agent for mapping regional hypoxia [21]. Because 18FFMISO partitions nearly equally between octanol and water, normoxic tissues have tissue-to-blood ratio (T/B) pixel values of almost 1.0. When the O2 supply is adequate, most tissues have relatively similar levels of 18F as in the blood. The hypoxic part of a tumor can be characterized by the maximum T/B value or by the total number of pixels with T/B greater than same threshold. 18F-FMISO PET could identify hypoxic tissue that is heterogeneously distributed within human tumors and can help to facilitate image-directed radiotherapy. 18F-FMISO imaging has also been used to identify postradiotherapy tumor recurrence by differential uptake of tracer. A significant correlation was found between hypoxic tissue identified by 18F-FMISO and both pimonida-

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zole and CA-IX, detected by immunohistochemical staining techniques. Several other compounds have also been evaluated as imaging agents [21]. 18F-fluoro-erythro-nitroimidazole (18F-FETNIM) has been evaluated in HNC. A derivative that is more hydrophilic than 18F-FMISO, 18F-fluoro-azomycin-arabinofuranoside (18F-FAZA), has been shown to be promising for clinical use, as it is the 18F-fluoro-etanidazole (18F-FETA) and EF5. An alternative PET agent for hypoxia is based on a metal complex of radioactive copper with ATSM, diacetyl-bis(N4-methylthiosemicarbazone) [21]. Dithiosemicarbazones have antitumor properties that are enhanced when they are complexed with Cu(II). Because there are several advantageous imaging radionuclides of copper, this has led several laboratories to develop substituted ligands of dithiosemicarbazones as potential imaging agents. Cu-ATSM uptake is more rapid than 18F-FMISO uptake, and the reported hypoxic to normoxic ratio is greater. One concern is that, because of its lipophilicity, the early uptake and washout of Cu-ATSM is probably influenced by regional blood flow, which is a major confounder with hypoxia [21]. Nevertheless, Cu-ATSM is finding a useful role in several clinical settings.

Hypoxia and Clinical Outcomes in Head and Neck Cancers Hypoxia appears to be prognostic for outcome in HNC, with data suggesting that hypoxia is prognostic for survival and local control. Several studies have shown that low pO2 in tumor, defined by either the median value or the hypoxic fraction, correlated with treatment outcomes in HNC patients treated with RT or chemoradiotherapy [22–24]. Brizel et  al. [24] reported 63 HNC patients with pretreatment tumor oxygen assessment, including primary site and lymph nodes. Hypoxia (tumor median pO2, 10  mmHg) adversely affected 2  year local–regional control, diseasefree survival, and overall survival (35% vs. 83%). It was also found that tumor pO2 predicted for pathologically persistent neck nodes in patients undergoing a neck dissection for clinical N2–3 necks after chemoradiation treatment [25]. In another study by Terris [26], only a small number of patients were assessed and hypoxia did not appear to be a prognostic factor. A multicenter study by Nordsmark et al. [27] involving 397 patients with HNC provided further evidence that tumor pO2 was an independent predictor for survival and tumor hypoxia was associated with a poor prognosis in patients with advanced HNC following primary radiotherapy. In HNC, hypoxia not only predicts for diseasefree survival and overall survival but also for local control, suggesting hypoxia-induced radiation resistance as an important factor for local failure.

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The prognostic impact of HIF-1a and HIF-2a expression has been the subject of numerous studies [28–31]. High HIF-1 expression has been correlated with a poorer survival in HNC treated with radiation with or without chemotherapy [28, 32]. Similar trends are observed in nasopharyngeal tumors [33]. Koukourakis et al. [28] assessed the expression of HIF-1a and HIF-2a in 75 cancer specimens from patients with HNSCC treated with concurrent chemoradiotherapy. HIF-1a and HIF-2a overexpression were shown in 52 and 33% of cancer samples, respectively. Bone/cartilage involvement was more frequent in tumors with high HIF-1a expression. HIF-1a and HIF-2a overexpression were significantly associated with high microvessel density and with VEGF expression. High levels of HIF-1a and HIF-2a expression were associated with incomplete response to chemoradiation, poor local relapse-free survival, and poor overall survival. HIF-2a expression was an independent prognostic factor in multivariate models. Aebersold et al. [32] explored the predictive potential of HIF-1a expression in 98 patients with oropharyngeal cancer treated by curative radiation therapy in which 94% of the primary tumors showed overexpression of HIF-1a. The degree of HIF-1a immuno reactivity correlated inversely with both the rate of complete remission of the primary tumor and lymph node metastases as well as with local failure, and overall survival. Winter et al. [34] investigated the role of expression of HIF-1a and HIF-2a in a series of 151 patients who underwent surgery for HNSCC. High HIF-1a was expressed in 45 of 140 tumors (30%) and HIF-2a was expressed in 21 of 139 tumors (14%). HIF-1a alone was associated with a worse disease-free survival, and high HIF-1a/high HIF-2a expression was also an independent prognostic factor. The immunohistochemical detection of the HIF-1a target gene Glut-1 has been shown to be correlated with a poorer survival in HNC [35]. Oliver et  al. [36] investigated the relationship between Glut-1 expression and clinical outcome of a retrospective series of 54 cases of oral squamous cell carcinomas. There was a significant relationship between those tumors which demonstrated intense staining of Glut-1 and loco-regional recurrence. Kunkel et al. [37] analyzed retrospectively Glut-1 expression in 118 patients with oral squamous cell carcinoma. The survival rate of patients with a low Glut-1 labeling index was significantly longer compared with patients with a high Glut-1 labeling index (138  months vs. 60  months), and Glut-1 expression was found to be an independent prognostic marker. The second target gene of HIF-1a which has been extensively studied with regard to its prognostic significance is CA-IX [38]. As with HIF-1a and Glut-1, most studies found a negative impact of high CA-IX expression in patient with HNC. In one study by Koukourakis et al. [39], HIF-2a and CA-IX were assessed in a series of patients treated with radiotherapy in the frame of the continuous

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hyperfractionated accelerated radiotherapy (CHART) randomized trial (54 Gy in only 12 days compared with conventional radiotherapy, 66  Gy in 6.5  weeks). Both high levels of HIF-2a and CA-IX were correlated with loco-regional control and survival, suggesting the importance of tumor hypoxia in HNC. However, no benefit was found with the accelerated regimen in the group of hypoxic tumors. In another study [40], tumors with a nonhypoxic profile, as defined as low HIF-1a and low CA-IX expression had significantly better local control. A recent work by Overgaard et  al. [41] used another hypoxia-related marker, the serum level of OPN, in a randomized trial that compared patients’ radiotherapy with and without an hypoxic sensitizer (nimorazole). The patients who benefited the most from the hypoxic modification were in the group with high levels of serum OPN, strongly suggesting that measuring tumor hypoxia before radiotherapy help to individualize irradiation in a more rational way. Studies showing a prognostic significance of 2-nitroimidazole markers have also been reported for HNC [19].

Strategies to Overcome Hypoxia-Induced Treatment Resistance in Head and Neck Cancers Hyperbaric Oxygen Treatment The most straightforward strategy to overcome hypoxia is to administer oxygen at a pressure higher than room air (usually 3 atm), i.e., hyperbaric oxygen treatment. The largest clinical trial with hyperbaric oxygen has been conducted by the British Medical Research Council, which randomized 1,669 patients between radiotherapy with or without hyperbaric oxygen [42]. Hyperbaric oxygen significantly improved both survival and local control after radiotherapy for head-and-neck tumors and showed promising results in HNC patients. Some of the earliest work toward this end was done using hyperbaric oxygen to radiosensitize cervical [43] or HNC [44]. Though there was some initial success with this technique, recent studies have indicated that combining radiation with hyperbaric oxygen results in significant increase of normal tissue toxicities [45]. A metaanalysis of randomized trials suggests that the use of hyperbaric oxygen breathing during RT can improve local control by 10% and also improve 5-year survival for irradiated head and neck tumors, however, it has not gained general acceptance for clinical use due to inconsistent responses, safety issues, and the high cost for implementation and especially due to the increased incidence of severe radiation toxicity [46].

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Accelerated Radiotherapy with Carbogen and Nicotinamide Another promising approach to decrease hypoxia in HNC is to combine radiotherapy with both the vasodilator nicotinamide and carbogen breathing (95%O2, 5%CO2) to increase tumor pO2. This strategy, so-called accelerated radiotherapy with carbogen and nicotinamide (ARCON) has produced excellent 3-year local control rates >80% for locally advanced stage T3–4 laryngeal and oropharyngeal cancers in a phase II study [47]. Following this promising result, a large randomized phase III clinical trial testing the efficacy of ARCON in laryngeal cancer patients has been performed in the Netherlands and should be presented in 2009 [48].

Improving Hemoglobin with Erythropoietin Manipulation Early studies were also done using blood transfusion to increase the oxygen transport and, thereby, increase the tumor tissue pO2. Despite some initial success [49] with this method transfusion failed to improve the local control in a randomized trial performed by the Danish Head and Neck Cancer (DAHANCA) group. Recently, blood transfusion has been supplanted by the administration of erythropoiesis-stimulating factors. Unfortunately, the combination of erythropoietin and radiotherapy is proved to be detrimental in several large randomized studies. Anemia is associated with a poorer outcome in patients treated with radiotherapy [50], possibly because it leads to low oxygen levels in tumors. Correction of anemia has been suggested to reverse this hemoglobin effect, thereby improving cancer control [51]. Recombinant human erythropoietin (EPO) can correct anemia and improve the quality of life in anemic patients with cancer. A phase III trial (ENHANCE study, 351 patients) was conducted to address the question whether anemia correction with erythropoietin could improve the outcome of curative radiotherapy among patients with HNSCC [52]. It showed that EPO corrected anemia, but tumor control, survival, and disease control rates were significantly worse when using EPO. This detrimental effect associated with EPO, when combined with RT in HNSCC was confirmed by the results of RTOG 99-03 [53] and DAHANCA-10 randomized studies (14th European Cancer Conference ECCO). In this later study, in a series of 515 patients eligible for analysis, a significantly poorer locoregional control rates was observed for the patients who received erythropoietin compared to the control group in HNSCC patients treated with radiotherapy. However, the target hemoglobin range in that study was 14.0–15.5 g/dl, which is beyond the optimal range for tumor oxygenation.

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The reason of the observed negative effect of EPO on tumor control could be that tumor oxygenation is decreased by both anemia and inappropriately high-hemoglobin levels. The latter are associated with an increased blood viscosity and a drop in nutritive perfusion. Hemoglobin concentrations between 12 and 14 g/dl could be optimal for maximum tumor oxygenation [51]. Thus, it is important to keep the hemoglobin concentrations within this range during radiotherapy. In addition, a retrospective analysis of a subset of patients from the ENHANCE study suggested that the expression of erythropoietin receptors on cancer cells can play an important role in HNSCC patients receiving erythropoietin during radiotherapy [54]. Loco-regional progression-free survival was substantially poorer if erythropoietin was administered to patients positive for the receptor expression compared with placebo, however, erythropoietin did not impair outcome in receptor-negative patients. Given these results, the use of EPO in HNC patients should not be considered outside controlled clinical trials [55].

Hypoxic Cell Radiosensitizers: Nitroimidazoles A widely investigated hypoxia-targeted strategy is to use electron-affinic drugs (nitroimidazoles) to sensitize tumors to the effect of radiation. Xenograft studies showed significant radiosensitization with nitroimidazole compounds in tumors without enhancing normal tissue toxicity. These encouraging results led to the realization of several of clinical trials exploring the clinical radiosensitizing potential of misonidazole in the late 1970s. However, the results of these clinical trials have been generally disappointing. One of the possible factor to explain the failure of misonidazole to provide significant advantage is the low plasma concentrations achievable with the permitted dose of this neurotoxic drug. Nevertheless, some was seen in one randomized trial. In the DAHANCA 2 trial [56], 626 patients with head and neck carcinoma were randomized to two different split-course radiation regimens and given either misonidazole or placebo during the initial 4  weeks of treatment. Overall, the misonidazole-treated group did not have a significantly better control rate than the placebo group. However, some benefit was found in patients with pharynx carcinomas. Misonidazole induced significant peripheral neuropathy in 26% of the treated patients, whereas other drug-related side effects were minimal. In the DAHANCA 5 trial [57], a less toxic nitroimidazole compound, nimorazole (Naxogin®), was used. Four hundred and twenty-two patients with carcinoma of the supraglottic larynx and pharynx were randomized to receive nimorazole or placebo, in association with conventional primary radiotherapy. With a median follow-up of 112  months, the nimorazole group showed a significantly

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better loco-regional control rate than the placebo group and a lower cancer-related death rate, without increasing the major side-effects.

Hypoxic Cell Cytotoxin: Bioreductive Drugs Bioreductive agents can selectively kill hypoxic cells. The first bioreductive drug used in clinical trials was mitomycin-C [58]. Haffty et al. [59] showed that the addition of mitomycin-C to RT resulted in statistically significant improvement in loco-regional control and cause-specific survival in HNC. Another study by Dobrowsky et al. [60, 61] comparing conventional fractionated RT to the Vienna continuous hyperfractionated accelerated RT regimen (V-CHART) or to V-CHART plus mitomycin-C showed that the best survival and loco-regional control rates were observed for the V-CHART and mitomycin-C group. However, the use of mitomycin-C is limited by its significant toxicity making it unlikely to be the ideal drug for exploiting tumor hypoxia. Recently, a new approach to tumor hypoxia has been developed using drugs that are preferentially cytotoxic to hypoxic cells [4], such as tirapazamine (TPZ). Preclinical studies have demonstrated that TPZ results in potentiation of both radiation and CDDP cytotoxicity [62]. In a phase I trial of TPZ, CDDP, and radiation (TPZ/CIS), impressive results were seen in locally advanced HNSCC [63]. This drug was then further evaluated in a randomized phase II trial TransTasman Radiation Oncology Group (TROG) 98.02 [64], 122 previously untreated advanced HNSCC patients were randomized to receive RT concurrently with either CDDP plus TPZ (TPZ/CIS), or CDDP and 5-FU. The striking observation of this study was that tumor control probability was strongly related to the pretreatment level of hypoxia, as measured by PET misonidazole. Hypoxic tumors treated with tirapazamine had an excellent control rate (>90%) while hypoxic tumors receiving 5-FU instead of tirapazamine had a very poor control rate (<25%) [65]. On the other hand, Rischin et al. reported results of a phase III trial HeadSTART of TROG during ASCO 2008. Eight hundred and sixty-one patients with previously untreated Stage III or IV HNSCC were randomized to receive RT concurrently with either CDDP (100  mg/m2 every 3  weeks) or CDDP (75  mg/m2 every 3 weeks + tirapazamine). No benefit was found due to the addition of TPZ to CT–RT in the absence of selection for the presence of hypoxia. All together, these two randomized studies suggest that a key issue in this area is to detect hypoxia and adapt the treatment to the characteristics of each individual tumor. In another phase II trial [66], 62 patients with lymph node-positive, resectable, stage IV HNSCC were randomized

10  Hypoxia in Head and Neck Cancers: Clinical Relevance and Treatment

to receive either two cycles of induction chemotherapy (TPZ, cisplatin, and 5-FU) followed by simultaneous chemoradiotherapy (TPZ, cisplatin, and 5-FU) or to receive the same regimen without TPZ. Patients who did not achieve a complete response at 50 Gy underwent surgical treatment. The addition of TPZ increased hematologic toxicity but did not improve outcomes in the small series of patients with resectable HNSCC.

Micro-Environment-Vascular Normalization Jain [67] has proposed the concept of normalization of tumor vasculature through antiangiogenesis and antivasculature targeted therapy [68]. Owing to high levels of proangiogenic molecules produced locally, such as VEGF, tumors become hypervascular, but the vessels are leaky and the blood flow is spatially and temporally heterogeneous. This leads to increased interstitial fluid pressure (IFP) and focal hypoxia, creating barriers to delivery and efficacy of therapeutics. The proposed mechanism of action of the VEGF-specific inhibitors, such as bevacizumab and sorafenib, is the inhibition of new-vessel formation and killing of immature tumor vessels, transient normalization of the remaining vasculature by decrease in macromolecular permeability (and thus the IFP) and hypoxia, and improvement of blood perfusion. The lowered IFP can lead to improved delivery of chemotherapeutics and molecularly targeted agents; the improved oxygenation sensitizes cancer cells to cytotoxic therapeutics and reduces the selection of more-malignant phenotype; and, finally, increased cellular proliferation around normalized vessels might increase the cytotoxicity of chemotherapeutics [69]. Normalization of the vasculature might also benefit the direct killing of cancer cells by bevacizumab, in synergy with the chemotherapeutics. Combined effects of bevacizumab with erlotinib and irradiation have been observed using a preclinical study on a HNC orthotopic model [70]. A phase I dose escalation study [71] has been conducted to evaluate the combination of bevacizumab and chemoradiotherapy (5-FU, hydroxyurea, radiation) in a series of 44 poor-prognosis HNC patients. Bevacizumab was integrated with chemoradiotherapy at a dose of 10  mg/m2 every 2  weeks. Some fistula formation/ tissue necrosis were observed that could be bevacizumabrelated. Erlotinib and bevacizumab combination has been investigated in 58 patients with recurrent or metastatic HNSCC in a phase I/II study [72]. The most common side effects of any grade were rash and diarrhea. A few patients could have benefit from this approach especially when the ratio of tumor-cell phosphorylated VEGF receptor-2 (pVEGFR2) over total VEGFR2 and endothelial-cell pEGFR

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over total EGFR in pretreatment biopsies were associated with complete response. A phase II trial of sorafenib has been conducted in a small series of 27 patients with recurrent or metastatic squamous cell carcinoma of the head and neck or nasopharyngeal carcinoma. Sorafenib was well tolerated with a few grade 3 and no grade 4 toxicities but had modest anticancer activity comparable to monotherapy with other targeted agents in this group of patients [73].

Targeting HIF-1 Given the role of HIF-1a in response to hypoxia, there is a major interest to develop specific HIF-1 inhibition. In xenograft assays, manipulation of HIF-1 activity by genetic or pharmacological means has marked effects on tumor growth along with some effects on angiogenesis, glucose metabolism, and/or cell survival [74]. Topotecan, a topoisomerase I poison that reversibly binds to and stabilizes the topoisomerase I enzyme, inhibited HIF-1 protein translation by a proteasome- and DNA damageindependent mechanism. Currently, topotecan is being tested in a clinical trial at the National Cancer Institute for its ability to inhibit HIF-1a protein expression and function in patients with advanced malignancies refractory to standard therapy [74]. Inhibitors of several upstream signaling pathways of HIF-1, such as EGFR and mTOR, have been extensively investigated in clinical trials in these recent years [7]. The mTOR inhibitors (everolimus and temsirolimus) that can suppress mTOR-dependent HIF-1 translation, and EGFR inhibitors (gefitinib, erlotinib) or antibodies (cetuximab, panitumumab) could inhibit HIF-1 induction by EGFRdependent pathways [8]. Hsp90 is a molecular chaperone associated with a number of proteins, which include transcription factors (AhR, glucocorticoid receptor, p53) and signaling kinases (Akt, ErbB2, Raf-1, v-Src), and ensures the proper conformation, localization, and function of these client proteins. Hsp90 inhibitors were found to induce ubiquitination and proteasomemediated degradation of HIF-1a in a VHL-independent fashion, under both normoxic and hypoxic conditions [74]. Histone deacetylase (HDAC) inhibitors have also been tested recently [74]. The dynamic process of reversible acetylation of the lysine residue of histone, and nonhistone proteins is controlled by HDAC and histone acetyltransferases. Acetylation of histone proteins is important for DNA chromatin conformation and regulation of gene expression. Acetylation of nonhistone proteins has been implicated in protein stability and function and direct acetylation of HIF1a has been suggested.

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PET-Based Intensity-Modulated Radiotherapy Image fusion techniques and the use of intensity-modulated and image-guided radiotherapy can allow to delineate hypoxic radioresistant subtarget volumes for delivering a partial tumor boost. PET could detect hypoxia in tumors and a higher dose could be given to the hypoxic areas, using intensity-modulated radiotherapy (IMRT). The MSKCC experience with microboosts on hypoxic areas up to 100 Gy. This approach requires that PET imaging be sensitive and specific enough to image hypoxia. In this framework, a validation of PET imaging used for adaptive radiotherapy was undertaken in animal models by comparing small-animal PET images (2.7 mm resolution) with autoradiography (AR) (100 mm resolution) in various tumors under various physiological situations [75]. Discrepancies were found between the PET images and the underlying microscopic reality represented by AR images. These differences, attributed to the finite resolution of PET, were important when considering small regions of the tumors. Dose painting based on PET images should be carefully considered and should take these limitations into account. The feasibility of a Cu-ATSM-guided IMRT approach through coregistering hypoxia (60)Cu-ATSM PET to the corresponding CT images for IMRT planning has been reported in HNC patients [76]. Radiation dose to the hGTV could be escalated without compromising normal tissue sparing (parotid glands and spinal cord). The plan delivered 80  Gy in 35 fractions to the ATSM-avid tumor subvolume and the GTV simultaneously receives 70 Gy in 35 fractions while more than one-half of the parotid glands were spared to less than 30 Gy. Thorwarth et  al. [77] investigated the feasibility of dif­ ferent hypoxia dose painting strategies in radiotherapy of 13 head and neck cancer patients. For each patient, three different treatment plans were created: a conventional IMRT plan, an additional uniform dose escalation (uniDE) of 10% to the fluorodeoxyglucose (FDG)-positive volume, and a plan in which dose painting by numbers (DPBN) was implemented. DPBN was realized according to a map of dose-escalation factors calculated from dynamic 18F-FMISO PET data. For DPBN, the prescriptions could be fulfilled in larger regions of the target than for uniDE. DPBN seems to result in higher benefits for the patients regarding tumor control probability. If hypoxia could be adequately quantified with a simple imaging technique such as FMISO positron emission tomography, DPBN in head-and-neck cancer could substantially increase tumor control. Lee et al. reported the results from a prospective study of pre-/midtreatment 18F-FMISO PET scans in a series of locoregionally advanced HNC patients treated with concomitant chemotherapy and IMRT [78]. Each patient underwent four

Y. Tao and J. Bourhis

PET scans: one pretreatment FDG PET/CT scan, two pretreatment 18F-FMISO PET/CT scans, and a final 18F-FMISO PET (midtreatment) scan performed 4  weeks after the start of chemoradiotherapy. An heterogeneous distribution of 18F-FMISO was noted in the primary and/or nodal disease in 90% of the patients. The positive 18F-FMISO findings of the midtreatment PET scan was not correlated with patient outcome. Another study has evaluated the influence of changes in tumor hypoxia on the efficacy of IMRT dose painting, according to serial 18F-FMISO PET imaging [79]. Seven patients with HNC were imaged twice with FMISO PET, separated by 3 days, before radiotherapy. IMRT plans were designed, on the basis of the first FMISO scan, to deliver a boost dose of 14 Gy to the hypoxic volume, in addition to the 70-Gy prescription dose. The changes in spatial distribution of tumor hypoxia, as detected in serial FMISO PET imaging added some complexity to define an adequate, coverage of hypoxic tumor volumes achievable by dose-painting IMRT and, dose painting potentially increased the EUD of the hypoxic volumes.

Other Methods Hyperfractionation radiotherapy (HFRT) [80] was designed to improve radiotherapy effectiveness by delivering two to three fractions daily with a reduced dose per fraction, which may reduce late toxicity despite an increased total dose. In addition, hyperfractionation could induce reoxygenation and its use was associated with an 8% improvement in survival at 5 years [81]. Other radiotherapy techniques can be of interest to overcome tumor hypoxia, such as high linear-energy transfer (LET) radiation which is less oxygen dependent. For example, carbon ions could be used to decrease the radiation resistance induced by hypoxia, and is currently under investigation. In conclusion, tumor hypoxia continues to be a therapeutic challenge in HNC. Nonetheless, the prospect of reducing its impact is looking brighter with improved ability of detecting hypoxia and better understanding of its molecular targets for therapeutic exploitation. Testing new leads from the laboratory will require clinical trials with innovative designs that incorporate serial novel noninvasive surrogate end points for hypoxia, such as molecular makers or imaging methods.

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178 43. Dische S, Anderson PJ, Sealy R, et al. Carcinoma of the cervix – anaemia, radiotherapy and hyperbaric oxygen. Br J Radiol. 1983;56:251–5. 44. Henk JM, Kunkler PB, Smith CW. Radiotherapy and hyperbaric oxygen in head and neck cancer. Final report of first controlled clinical trial. Lancet. 1977;2:101–3. 45. Bennett M, Feldmeier J, Smee R et al. Hyperbaric oxygenation for tumour sensitisation to radiotherapy. Cochrane Database Syst Rev. 2005;4:CD005007. 46. Overgaard J, Horsman MR. Modification of hypoxia-induced radioresistance in tumors by the use of oxygen and sensitizers. Semin Radiat Oncol. 1996;6:10–21. 47. Kaanders JH, Pop LA, Marres HA, et al. ARCON: experience in 215 patients with advanced head-and-neck cancer. Int J Radiat Oncol Biol Phys. 2002;52:769–78. 48. Kaanders JH, Bussink J, van der Kogel AJ. ARCON: a novel biologybased approach in radiotherapy. Lancet Oncol. 2002;3:728–37. 49. Evans JC, Bergsjo P. The influence of anemia on the results of radiotherapy in carcinoma of the cervix. Radiology. 1965;84:709–17. 50. Denis F, Garaud P, Bardet E, et al. Final results of the 94-01 French Head and Neck Oncology and Radiotherapy Group randomized trial comparing radiotherapy alone with concomitant radiochemotherapy in advanced-stage oropharynx carcinoma. J Clin Oncol. 2004;22:69–76. 51. Rades D. Erythropoietin administration during radiotherapy in anaemic head-and-neck cancer patients: is it still a reasonable option or too dangerous? Oral Oncol. 2009;45:91–3. 52. Henke M, Laszig R, Rube C, et al. Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial. Lancet. 2003;362:1255–60. 53. Machtay M, Pajak TF, Suntharalingam M, et al. Radiotherapy with or without erythropoietin for anemic patients with head and neck cancer: a randomized trial of the Radiation Therapy Onco-logy Group (RTOG 99-03). Int J Radiat Oncol Biol Phys. 2007;69:1008–17. 54 Henke M, Mattern D, Pepe M, et al. Do erythropoietin receptors on cancer cells explain unexpected clinical findings? J Clin Oncol. 2006;24:4708–13. 55. Bokemeyer C, Aapro MS, Courdi A, et al. EORTC guidelines for the use of erythropoietic proteins in anaemic patients with cancer: 2006 update. Eur J Cancer. 2007;43:258–70. 56. Overgaard J, Hansen HS, Andersen AP, et al. Misonidazole combined with split-course radiotherapy in the treatment of invasive carcinoma of larynx and pharynx: report from the DAHANCA 2 study. Int J Radiat Oncol Biol Phys. 1989;16:1065–8. 57. Overgaard J, Hansen HS, Overgaard M, et al. A randomized double-blind phase III study of nimorazole as a hypoxic radiosensitizer of primary radiotherapy in supraglottic larynx and pharynx carcinoma. Results of the Danish Head and Neck Cancer Study (DAHANCA) Protocol 5-85. Radiother Oncol. 1998;46:135–46. 58. De Ridder M, Van Esch G, Engels B, et  al. Hypoxic tumor cell radiosensitization: role of the iNOS/NO pathway. Bull Cancer. 2008;95:282–91. 59. Haffty BG, Son YH, Papac R, et al. Chemotherapy as an adjunct to radiation in the treatment of squamous cell carcinoma of the head and neck: results of the Yale Mitomycin Randomized Trials. J Clin Oncol. 1007;15:268–76. 60. Dobrowsky W, Naude J, Widder J, et al. Continuous hyperfractionated accelerated radiotherapy with/without mitomycin C in head and neck cancer. Int J Radiat Oncol Biol Phys. 1998;42:803–6. 61. Dobrowsky W, Naude J. Continuous hyperfractionated accelerated radiotherapy with/without mitomycin C in head and neck cancers. Radiother Oncol. 2000;57:119–24. 62. Gandara DR, Lara Jr PN, Goldberg Z, et al. Tirapazamine: prototype for a novel class of therapeutic agents targeting tumor hypoxia. Semin Oncol. 2002;29:102–9. 63. Rischin D, Peters L, Hicks R, et al. Phase I trial of concurrent tirapazamine, cisplatin, and radiotherapy in patients with advanced head and neck cancer. J Clin Oncol. 2001;19:535–42.

Y. Tao and J. Bourhis 64. Rischin D, Peters L, Fisher R, et al. Tirapazamine, Cisplatin, and Radiation versus Fluorouracil, Cisplatin, and Radiation in patients with locally advanced head and neck cancer: a randomized phase II trial of the Trans-Tasman Radiation Oncology Group (TROG 98.02). J Clin Oncol. 2005;23:79–87. 65. Rischin D, Hicks RJ, Fisher R, et  al. Prognostic significance of [18F]-misonidazole positron emission tomography-detected tumor hypoxia in patients with advanced head and neck cancer randomly assigned to chemoradiation with or without tirapazamine: a substudy of Trans-Tasman Radiation Oncology Group Study 98.02. J Clin Oncol. 2006;24:2098–104. 66. Le QT, Taira A, Budenz S, et al. Mature results from a randomized Phase II trial of cisplatin plus 5-fluorouracil and radiothe-rapy with or without tirapazamine in patients with resectable Stage IV head and neck squamous cell carcinomas. Cancer. 2006;106:1940–9. 67. Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science. 2005;307:58–62. 68. Winkler F, Kozin SV, Tong RT, et al. Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases. Cancer Cell. 2004;6:553–63. 69. Jain RK, Duda DG, Clark JW, et al. Lessons from phase III clinical trials on anti-VEGF therapy for cancer. Nat Clin Pract Oncol. 2006;3:24–40. 70. Bozec A, Sudaka A, Fischel JL, et al. Combined effects of bevacizumab with erlotinib and irradiation: a preclinical study on a head and neck cancer orthotopic model. Br J Cancer. 2008;99:93–9. 71. Seiwert TY, Haraf DJ, Cohen EE, et  al. Phase I study of bevacizumab added to fluorouracil- and hydroxyurea-based concomitant chemoradiotherapy for poor-prognosis head and neck cancer. J Clin Oncol. 2008;26:1732–41. 72. Cohen EE, Davis DW, Karrison TG, et  al. Erlotinib and bevacizumab in patients with recurrent or metastatic squamous-cell carcinoma of the head and neck: a phase I/II study. Lancet Oncol. 2009;10:247–57. 73. Elser C, Siu LL, Winquist E, et  al. Phase II trial of sorafenib in patients with recurrent or metastatic squamous cell carcinoma of the head and neck or nasopharyngeal carcinoma. J Clin Oncol. 2007;25:3766–73. 74. Melillo G. Targeting hypoxia cell signaling for cancer therapy. Cancer Metastasis Rev. 2007;26:341–52. 75. Christian N, Lee JA, Bol A, et al. The limitation of PET imaging for biological adaptive-IMRT assessed in animal models. Radiother Oncol. 2009;91:101–6. 76. Chao KS, Bosch WR, Mutic S, et al. A novel approach to overcome hypoxic tumor resistance: Cu-ATSM-guided intensitymodulated radiation therapy. Int J Radiat Oncol Biol Phys. 2001;49:1171–82. 77. Thorwarth D, Eschmann SM, Paulsen F, et al. Hypoxia dose painting by numbers: a planning study. Int J Radiat Oncol Biol Phys. 2007;68:291–300. 78. Lee N, Nehmeh S, Schoder H, et  al. Prospective trial incorporating pre-/mid-treatment [(18)F]-misonidazole positron emission tomography for head-and-neck cancer patients undergoing concurrent chemoradiotherapy. Int J Radiat Oncol Biol Phys. 2009;75:101–8. 79. Lin Z, Mechalakos J, Nehmeh S, et al. The influence of changes in tumor hypoxia on dose-painting treatment plans based on 18F-FMISO positron emission tomography. Int J Radiat Oncol Biol Phys. 2008;70:1219–28. 80. Bourhis J, Lapeyre M, Tortochaux J, et  al. Phase III randomized trial of very accelerated radiation therapy compared with conventional radiation therapy in squamous cell head and neck cancer: a GORTEC trial. J Clin Oncol. 2006;24:2873–8. 81. Bourhis J, Overgaard J, Audry H, et al. Hyperfractionated or accelerated radiotherapy in head and neck cancer: a meta-analysis. Lancet. 2006;368:843–54.

Chapter 11

Translational Research in Head and Neck Oncology David S. Yoo and David M. Brizel

Abstract  Translational research in head and neck oncology has evolved dramatically. Ongoing discoveries in basic mechanisms of cancer biology and technological advances in both diagnostic imaging and radiation delivery have enhanced the ability to improve treatment outcomes. The overarching goal for all translational research should be to enlarge the armamentarium from which clinicians can rationally select the most appropriate options for individual patients in ways that maximize therapeutic benefit and minimize toxicity. Focusing on this goal will become more critical as the health care system deals with external economic, social, and political pressures and forces that will affect both bench and bedside. As these concerns encroach on the translational process, it is imperative to recognize that the research itself is best equipped to address them – more efficacious treatments, improved patient selection, decreased toxicity. What also should not be lost in translation is the unpredictable and occasional serendipitous nature of research. Two cornerstones of head and neck cancer therapy, cisplatin [1, 2] and cetuximab, owe their existence to chance and fate. Meanwhile, the compelling story of tumor hypoxia has yet to result in any new additions to the therapeutic arsenal. This chapter will explore the meaning of translational research means, identify potential pitfalls on the horizon, and highlight common themes and new avenues of research using specific examples from both the head and neck and general oncology literature. Keywords  Translational research • Oncology • Targeted therapy • Cetuximab

Introduction Translational research is not unlike world peace, the meaning of which depends upon whom is asked. But it sounds great, and everyone is for it. Unfortunately, success can be elusive, with many setbacks along the way. Progress requires seeking and forging of new relationships, many times between seemingly unrelated and disparate camps that speak different languages. The concept of translational research in oncology evokes images of a bridge, spanning and connecting the separate worlds of basic bench research and clinical bedside investigation and treatment. Cellular and molecular discoveries in the laboratory yield clues to underlying mechanisms of disease, identifying novel targets for therapeutic intervention that ultimately improve cancer patient outcomes. The National Cancer Institute expands on this concept, defining translational research as that which “transforms scientific discoveries that arise from laboratory, clinical, or population studies into clinical applications to reduce cancer incidence, morbidity, and mortality” [3]. The discipline of head and neck oncology possesses a strong history of translational research and continues to expand and build upon its foundation of scientific discoveries. Several chapters in this textbook are singularly devoted to epidemiology, genetics, virology, proteomics, predictors and prognosticators, hypoxia, targeted therapies, and functional imaging. Other chapters discuss preclinical models and phase I study methodology. Translational research links these topics together and is ultimately responsible for writing and shaping the current and future chapters on patient management and evidence-based practice.

Roadblocks D.M. Brizel (*) Department of Radiation Oncology, Duke University Medical Center, Morris Building, Science Drive, Durham, NC 27710, USA e-mail: [email protected]

One of the ironic aspects of cancer research today is that the sheer avalanche of data and knowledge generated may overwhelm the ability to ask the most appropriate clinical

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questions. When the haystack is filled with needles, finding one gives way to the more challenging task of finding the right one. For example, at least 12 different agents target the epidermal growth factor receptor (EGFR) alone [4]. There are four downstream pathways associated with EGFR, and the number of potential therapeutic strategies to shepherd through from conception to daily practice expands geometrically along each signaling cascade [5]. The danger then becomes one of seeing a promising new treatment get lost in the translation. The Clinical Research Roundtable at the Institute of Medicine (IOM) in their special communication to JAMA in 2003 highlighted an example of one of the dilemmas in translational research [6]. The IOM, comprised of individuals from the fields of nursing, medicine, basic science, public health, medical informatics, insurance companies, industry, and private foundations, described two translational roadblocks that “impede efforts to apply science to better human health in an expeditious fashion.” The first exists when trying to convert basic in  vitro and in  vivo laboratory discoveries into novel interventions for human studies. The second occurs in the process of applying the results of these human studies and attempting to integrate them into everyday clinical practice and decision-making. The culprits deemed to be responsible for both blocks including insufficient funding, insufficient infrastructure, lack of qualified personnel, lack of career incentives, and a dearth of willing research subjects. Much of the emphasis and funding in medical research to date has been placed on trying to overcome the first block. Novel therapeutics and new diagnostic modalities generate great excitement and enthusiasm, translating well not only within the medical profession, but also to the general public as well. Many are now concerned, however, that the second translational block constitutes the greatest bottleneck and is most detrimental to the health outcomes of everyday patients. More people, it has been argued, can be better served by focusing on the appropriate delivery of already proven treatment strategies rather than inventing new ones [7]. For example, the expenditure of effort to develop new and incrementally more efficacious statins or antiplatelet drugs contributes less to the overall societal health than using those same resources to ensure delivery of already available drugs to all eligible patients [8]. US health care expenditures in 2007 totaled $2.4 trillion, nearly 17% of the gross domestic product (GDP) [9]. Current projections are for this sum to increase to 20% of GDP by 2017. Historically, approximately 5% of this spending has been related to cancer therapy, although this percentage is also expected to rise with the aging US population and the adoption of newer, more expensive technologies and therapies [10]. How much should be spent and what level of care it should buy will require national debate and political intervention, meaning the probability of a rational solution is low.

D.S. Yoo and D.M. Brizel

The growing awareness of the extent to which new cancer treatments contribute to the escalating costs of health care has resulted in urgent calls to police within the oncology community before outside government agencies are mandated to do so. Such external intervention could set up more translational blocks, likely with less precision and more regulation. A recent report from the NIH reviewed four molecular targeted agents – cetuximab, bevacizumab, erlotinib, sorafenib – pinnacles of the translational research effort and compared their “purported” benefits and estimated costs [11]. They highlighted the recent multinational phase III FLEX (First-Line ErbituX) study comparing platinum-based chemotherapy with or without cetuximab as first-line therapy in EGFR-overexpressing nonsmall cell lung cancer patients with either wet stage IIIB or stage IV disease [12]. Patients randomized to the cetuximab arm received a loading dose of 400 mg/m2, followed by weekly doses of 250 mg/m2 concurrent with up to six cycles of chemotherapy and continuing weekly until disease progression or unacceptable toxicity. The primary endpoint was achieved with a statistically significant increase in median survival from 10.1 to 11.3 months with the addition of cetuximab. Ten percent developed grade 3 acne-like skin toxicity. The cost of adding cetuximab to 18  weeks of chemotherapy (60 kg patient and $11.52 per mg of cetuximab) was $80,352 per patient [11]. Similarly, the addition of the small molecule tyrosine kinase inhibitor erlotinib to gemcitabine in advanced pancreatic cancer increased median survival by 10  days [13] for a cost of $15,752 [11]. Similar examples were presented for the use of bevacizumab in metastatic breast cancer [14] and sorafenib in renal cell carcinoma [15], emphasizing the tension that exists reconciling the costs of these therapies and their limited impacts on overall survival and/or quality of life. The EXTREME (ErbituX in first-line Treatment of REcurrent or MEtastatic head and neck cancer) trial had a very similar design to the FLEX study in lung cancer. In this trial, 442 patients with previously untreated recurrent or metastatic disease not amenable to local therapy were randomized to platinum and 5FU-based chemotherapy alone versus chemotherapy with weekly cetuximab [16]. Those patients with stable disease on concurrent therapy continued with weekly cetuximab until disease progression or unacceptable toxicity. The addition of cetuximab improved median survival from 7.4 to 10.1 months, along with improvements in progression-free survival and response rates. This increase represented a significant achievement in the recurrent/metastatic setting, the first intervention shown to improve survival in this population since cisplatin over 30 years ago [17]. However, this 2.7-month improvement in EXTREME may face further scrutiny, given the shot across the bow from the NIH regarding the results of FLEX. A typical patient in the USA with a body surface area of 2  mg/m2

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would have required 9,300 mg of cetuximab in 18 weeks in the experimental arm of the EXTREME trial at a cost of $107,136 based on 2008 data. Weekly treatment for 12 months in the setting of stable disease would have received 26,300  mg, which would have cost $302,976. Neither a privately run nor a publicly administered health care system can sustain this level of expense. A potential doomsday scenario for translational research could result if insurance companies and/or governments decide to offer patients a fraction of that cost to NOT take therapy. The American Society of Clinical Oncology published the initial deliberations of its Cost of Care Task Force focusing on the perspectives of the different stakeholders in the oncology community – patients, industry, payers, and physicians – and highlighted the need to “define the value of specific cancer interventions” [18]. Some advocate funding restraints on research studies which would place cost limits on experimental interventions depending on their potential survival advantages [11]. In the same vein, some industry stakeholders may decide that certain disease entities, including head and neck cancers, lack the necessary patient numbers and potential market share for allocation of their resources in support of clinical trials.

Common Themes The story of ICI 46,474, more commonly known as tamoxifen, is an instructive case study. This compound was first developed in the 1970s by Imperial Chemical Industries Ltd. Pharmaceuticals Division (now AstraZeneca) as a postcoital contraceptive [19]. The initial research that established tamoxifen as an antiestrogen capable of controlling hormonedependent tumors almost did not happen. At the time, the company did not see a financial incentive to market a drug used for a short period of time by a small number of metastatic breast cancer patients, most of who were getting the latest and most promising therapy, cytotoxic chemotherapy combinations. It took the threatened resignation of the Head of Research, serendipity and years of preclinical data before the antitumor activity of tamoxifen was established. Moreover, testing in humans was originally performed in patients with advanced metastatic disease. Although somewhat effective, it was not until tamoxifen was studied in an adjuvant setting that the large benefits in reducing recurrence and improving overall survival were seen in estrogen receptor positive patients [20]. As stated by Dr. Jordan, the man who helped translate tamoxifen into clinical practice, “the key to success was targeting women with the right tumor with the correct duration of treatment at the right stage.” The right woman, the right tumor, at the right stage – many parallels can be drawn from the tamoxifen story to the

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targeted agents of today. Cetuximab’s origins can be traced back to a woman born in the late nineteenth century. What she did for the first eight decades of her life is not known, but at the age of 85, her squamous cell carcinoma of the vulva was harvested and transformed into the immortalized cell line A431 [21]. Eleven years later in 1984, her cell line provided the substrate for the creation of murine monoclonal antibodies against the EGF receptors over-expressed along the cell surface [22]. In 1991, one of these antibodies, mAb 225, was successfully injected and studied in human subjects [23]. By 1995, the chimeric antibody C225, aka cetuximab, was developed to overcome the human antimouse antibody phenomenon that limited the clinical utility of mAb 225 [24]. Head and neck cancer patients with over-expression of EGFR were noted to have a poorer prognosis, providing the rationale for targeted therapy with C225 [25, 26]. Cetuximab has been utilized in a variety of different clinical scenarios since – as a single agent in advanced chemorefractory disease [27], with chemotherapy in the recurrent/metastatic setting [16, 28], with radiation therapy alone in locally advanced but nonmetastatic patients [29], and with concurrent chemoradiation [30]. In refractory patients, single-agent cetuximab showed a median survival of 178  days [27]. The results of EXTREME in previously untreated recurrent/metastatic patients were outlined earlier, showing an increase in median survival from 7.4 to 10.1 months [16]. The phase III pivotal trial from Bonner et  al., which compared radiation therapy alone in the definitive setting with or without cetuximab, showed significant improvements in both local control and survival, increasing median survival from 29.3 to 49 months and 3  year overall survival from 45 to 55% [29]. As with tamoxifen, the earlier utilization of cetuximab in the nonmetastatic and treatment-naïve setting demonstrated a more robust improvement in clinical outcomes. Building upon these findings, the RTOG has recently completed enrollment onto a phase III study evaluating whether the addition of cetuximab to definitive chemoradiation can further improve outcome. The fate of cetuximab and other novel therapeutic agents as they progress through various phases of development highlights several important themes for current and future translational research efforts. As the specificity of these agents toward their molecular targets increases, so too should the process of patient selection in order to optimally use them in various clinical scenarios. The keys to success require several interrelated questions to be addressed: who gets therapy, what agent(s) gets tested, whether to give or not give therapy, scheduling, and sequencing, where is the primary tumor located, and why did things work or not work? Limitations on resources and competition for study patients will prevent all of these questions from being asked. The head and neck oncology community will need to prioritize which ones are most important.

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Who Gets Treated The standard approach for new investigational agents that survive the preclinical gauntlet is to first test them in patients that have failed all known conventional therapies, initially for dose-limiting toxicities and safety and then for efficacy. An exciting and challenging avenue for research is now asking how improvements in outcomes in the recurrent and refractory setting translate in treatment-naïve patients. Are the additional months in median survival outback simply reshuffled upfront? Or are there true qualitative and quantitative improvements in survival, with more cures and less patients going on to require therapy for recurrent or metastatic disease? In head and neck cancer, the EXTREME and Bonner studies suggest the latter. This has not always been the case. In colorectal cancer, the addition of bevacizumab to irinotecan, bolus fluorouracil, and leucovorin in previously untreated metastatic patients resulted in a statistically significant improvement in survival (median duration 15.6 vs. 20.3 months, HR 0.66, p < 0.001) [31]. A similar benefit in overall survival was seen in a phase III ECOG study in patients with previously treated metastatic colorectal cancer. In this trial, the addition of bevacizumab to fluorouracil, leucovorin, and oxaliplatin (FOLFOX) improved median survival from 10.8 to 12.9  months compared to FOLFOX alone [32]. However, the survival benefits of adding bevacizumab to standard of care chemotherapy do not appear to automatically translate in the nonmetastatic setting. Preliminary results from NSABP C-08 showed no statistically significant improvement in disease-free survival with the addition of bevacizumab to FOLFOX in resected stage II–III colon cancer patients [33]. Another more ominous example is a phase III SWOG adjuvant lung cancer study. Patients received definitive concurrent thoracic chemoradiation and consolidation docetaxel chemotherapy with or without the addition of gefitinib, a small molecule EGFR tyrosine kinase inhibitor. Patients receiving gefitinib had a significant decrease in median survival (23 vs. 35  months) [34]. These findings further emphasize the importance that promising preclinical and early phase data for targeted agents must be validated in a rigorous phase III setting before they can be incorporated into widespread clinical practice. Even then, the translation of successful randomized phase III trials in the phase IV practice setting can encounter unexpected hazards. Cetuximab is associated with an approximate 3–4% incidence of grade 3–4 infusion reactions in the USA. However, in certain geographic locations, the rate of severe anaphylactic hypersensitivity-type reactions approaches 20–25% [35]. In an illustrative example of bedside-to-bench reverse translation, these reactions have been linked to preexisting IgE antibodies that cross-react to a

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galactose-alpha-1,3-galactose moiety that is tagged to the Fab portion of the mouse component of the cetuximab molecule during antibody production [36]. Moreover, preexisting IgE antibodies in the general population were found to be more prevalent in people from Tennessee, Arkansas, and North Carolina (20.8%) as opposed to northern California (6.1%) or Boston (0.6%). The potential increased risk for these severe reactions has limited the enthusiasm for and restricted utilization of cetuximab in pockets of the Southeast USA. It was perhaps serendipitous that C225 was developed in other parts of the country. Parallels may be drawn to trials examining the addition of concurrent chemotherapy to radiation in nasopharyngeal carcinoma (NPC), a tumor known for significant geographic variability with regards to histology and EBV status. Following the positive results of the Intergroup 0099 trial [37], studies were undertaken throughout Asia to determine whether the significant survival benefit seen in North American patients with a concurrent chemoradiation strategy translated to the endemic form of NPC found more predominantly in that part of the world. Three phase III trials from Taiwan, Singapore, and Hong Kong confirmed a survival benefit with concurrent chemoradiation versus radiation alone [38–40]. However, preliminary results from a fourth study with nonkeratinizing/undifferentiated histology patients from Hong Kong and Canada showed no survival benefit but increased acute and late toxicity with concurrent chemoradiation [41]. Whether regional or demographic differences in efficacy and/or toxicity will be discovered with targeted therapies remains to be seen. The question of who gets certain therapies is further complicated by the growing awareness of a likely causal association between certain subsets of head and neck cancers and the human papillomavirus (HPV) [42]. These doublestranded DNA viruses have survived millennia in the inhospitable terminally differentiated epithelia of higher level organisms, cleverly restarting their nondividing hosts’ replication machinery by inactivating both the p53 and pRb tumor suppressors. The first suggestion of HPV involvement in head and neck cancer came in 1983 based on histopathologic findings seen in a subset of oral squamous cell carcinomas similar to those caused by HPV in the uterine cervix [43]. Detection of high risk HPV16 DNA in tonsil cancer specimens came in 1990 [44]. Multiple retrospective series and a subsequent meta-analysis suggested that patients with HPV-positive oropharyngeal tumors had improved disease-free and overall survival, with a 28% reduced risk of death compared to HPV-negative patients [45]. The prognostic significance of HPV status was demonstrated prospectively in 96 patients from a phase II ECOG study examining an induction chemotherapy regimen followed by chemoradiation [46]. Patients with HPV-positive tumors had higher response rates to chemotherapy and chemoradiation, as well

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as a 2-year overall survival of 95% [95% CI = 87–100%] versus 62% [95% CI = 49–74%] for the HPV-negative patients. The improved outcomes and atypical presentations (younger age, female, lack of prior tobacco and alcohol use) of HPV-positive head and neck cancer patients suggest these tumors may represent a distinct clinical entity [47]. Given the potential for confounding of clinical trial results, future translational studies in head and neck cancer will likely need to stratify patients according to HPV status [48]. RTOG 0619, discussed below, includes stratification of oropharyngeal primary tumors by HPV status. Moreover, the excellent prognosis of HPV-positive patients has further implications regarding the future direction of treatment strategies that incorporate novel translational therapies. The question arises whether intensive concurrent regimens using radiation, chemotherapy, and molecular targeted agents are necessary for optimal tumor control in HPV-positive patients or are they just more toxic. Therefore, strategies for deintensification of therapy in this subset of patients, including radiation dose reduction and/or combining radiation with lower doses of cisplatin chemotherapy or with well-tolerated targeted agents in lieu of chemotherapy, will likely be emphasized in the near future.

What With more stratification and potential reclassification of HPVpositive patients into a separate disease entity, the already small pie of head and neck cancer eligible to participate in clinical trials could get sliced further, reducing the ability to definitively answer study questions. RTOG 9003, the largest trial in head and neck cancer, needed over 6 years to enroll 1,113 patients [49]. Already, the increasing number of investigational agents has likely outgrown the number of people available for enrollment in clinical trials and the resources available to conduct them. To date, the RTOG has opened four head and neck protocols with targeted agents. Three are closed to accrual, 0234 and 522 with cetuximab, and 0615 with bevacizumab in NPC. The open 0619 study examines the addition of vandetanib, a dual EGFR and VEGFR tyrosine kinase inhibitor, to cisplatin in high-risk postoperative patients with extracapsular extension and/or microscopic positive margins. During the initial conception of 0619, the authors tallied the number of ongoing phase I/II studies in head and neck cancer with targeted agents, noting 32 trials involving cetuximab, gefitinib, erlotinib, panitumumab, celecoxib, bevacizumab, and lapatinib. The study of one agent at a time is challenging enough, with or without radiation, with or without chemotherapy. Another area of increasing interest involves targeting multiple signaling pathways at once, either with multiagent

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cocktails or more promiscuous inhibitors such as vandetanib. The rationale for this approach has been the limited clinical utility seen with single targeted agents alone and the redundancy of signaling pathways. Despite the fact that a majority of head and neck tumors have EGFR over-expression, cetuximab with radiation therapy still showed a 50% local recurrence rate in the Bonner trial [29]. This fact is not surprising, given the complexity of the molecular signaling pathways involved in the pathogenesis of head and neck cancers [50]. Preclinical studies have shown significant cross-talk, with both direct and indirect associations between the various signaling cascades, providing alternative routes to bypass inhibition of one pathway [51]. Already, the simultaneous inhibition of the EGFR and VEGF pathways with erlotinib and bevacizumab has been studied in the recurrent/metastatic setting, showing the combination was well tolerated and potentially more efficacious in a subset of patients with molecular evidence of activated pathways [52]. At Duke University, a phase I/II trial examining the use of erlotinib, bevacizumab, and concurrent cisplatin with hyperfractionated radiation therapy in treatment-naïve, locally advanced nonmetastatic patients has recently completed accrual. Median follow-up is 2  years, and the results have been promising, with only 2 of the 28 patients having had a local recurrence. The trial design has also incorporated companion studies with serial functional imaging scans and serum samples collected at time points before, during, and after the completion of therapy. The goal is to help identify potentially predictive and/or prognostic factors that correlate with treatment outcomes, improving the selection of patients for targeted therapies in the future. However, more is not always better. The Dutch CAIRO2 study in metastatic colorectal cancer found that the addition of cetuximab to capecitabine, oxaliplatin, and bevacizumab lead to a decrease in progression-free survival and quality of life [53]. The search for molecular rationales, including mechanisms of resistance, will require more bench research to help translate these unexpected bedside findings.

When Clearly, not every patient benefits from the administration of targeted therapies. Even with the potential for more dramatic clinical improvements in the definitive and nonmetastatic setting, it does not appear economically feasible to incorporate one or two (or more) targeted therapies into the treatment regimen of every patient that presents de novo with locally advanced head and neck cancer. Finding biomarkers and molecular assays that can reliably predict who might respond favorably to certain agents and when they should be utilized is a key emphasis of ongoing studies. In colorectal

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cancer, patients with EGFR expressing tumors and unresectable metastatic disease were randomized to FOLFIRI chemotherapy with or without cetuximab. Tumor KRAS gene mutation status was also examined. A progression-free survival benefit for cetuximab was limited to those patients with wild-type KRAS [54]. In the previously mentioned phase I/ II study examining erlotinib and bevacizumab in recurrent/ metastatic head and neck cancer, patients with increased phosphorylation of VEGFR in tumors and EGFR in endothelial cells were more likely to have complete responses [52]. Another study examining cisplatin and erlotinib in recurrent/ metastatic head and neck patients found a correlation between improved treatment response and high EGFR gene copy number [55]. More robust and clinically applicable prognostic and/or predictive tools will be identified and validated. In fact, given the current climate, research that results in the more judicious use of novel therapies is mission critical to the viability and support of future translational studies. The ability to identify responders versus nonresponders to targeted therapy early on in the treatment course would further improve patient selection and efficacy, providing guidance on when changes in therapy should be made. Recent trials with targeted agents have incorporated correlative studies with functional imaging modalities to noninvasively and serially assess the tumor microenvironment and monitor any possible treatment-related changes. Tools such as dynamic contrast-enhanced MRI (DCE-MRI) and PET-based assays attempt to capture novel information based on the underlying tumor biology, yielding potentially prognostic and predictive information to augment the anatomically based TNM staging system. For example, many antiangiogenic targeted agents exert their effects on tumor perfusion and vascular permeability, physiologic processes that can be quantitatively measured with DCE-MRI [56]. In breast cancer, early changes in tumor microvessel functionality as monitored by changes in DCE-MRI signaling predicted final clinical and pathologic response to neoadjuvant chemotherapy [57]. Other DCE-MRI parameters have also correlated with local control, diseasefree, and overall survival in multiple tumor sites, including lung, cervix, and head and neck [58–63].

How The question of how to optimally incorporate novel therapeutic agents in radiation-based treatment regimens is an active area of research. One limitation of the Bonner cetuximab trial that likely impacted widespread accrual and subsequent acceptance into clinical practice was the use of a control arm in the study that utilized radiation therapy alone in locally advanced patients. Based on the meta-analysis of Chemotherapy in Head and Neck Cancer (MACH-NC), which

D.S. Yoo and D.M. Brizel

examined updated patient data on 16,485 patients from 87 trials published between 1965 and 2000, the addition of chemotherapy to radiation provided an absolute benefit of 4.5% at 5 years with a hazard ratio of 0.88 [64]. This benefit was more pronounced (6.5% at 5 years, HR 0.81) with the concomitant use of chemotherapy and radiation as compared to induction or adjuvant strategies. The RTOG is addressing in two phase III trials whether the addition of targeted agents to current standards of care in both locally advanced and high-risk postoperative patients provides further benefit. RTOG 0522 asks whether cetuximab improves outcome when added to concurrent chemoradiation in the definitive setting while RTOG 0619, as described previously, is testing whether vandetanib improves upon combined modality therapy in high-risk postoperative patients. How novel targeted agents are incorporated into subsequent treatment regimens will be a critical area of ongoing research. Potential improvements in efficacy will need to be balanced against any increases seen in acute and late toxicity. In this context, tools to improve patient selection will play increasingly more important roles to optimally match treatment regimens of varying intensities to individual patients in order to optimize their therapeutic ratio. The addition of targeted agents to concurrent chemoradiation may represent the evolution of a new standard of care for patients with high-risk, poor prognosis disease. In other clinical scenarios, such as HPV-positive patients with better prognoses where chemotherapy may not be necessary or in elderly patients where the addition of chemotherapy may only increase toxicity, targeted agents may ultimately replace concurrent chemotherapy [65]. For example, the use of lapatinib with concurrent chemoradiation is being evaluated in locally advanced head and neck patients [66]. At the same time, others are examining lapatinib with radiation therapy alone in locally advanced patients who cannot tolerate chemoradiation [67]. One hypothesis-generating result from Bonner’s pivotal trial arises from the differences in survival seen between those patients who received cetuximab with altered fractionation versus conventional daily treatment schedules. Subset analyses showed that patients treated with concomitant boost regimens had a hazard ratio (HR) of 0.62 while the hyperfractionation group had a HR = 0.74. No difference in survival was seen in those patients who underwent conventional fractionation (HR = 1.01) [29]. This suggests that a trial design that utilized only conventional radiation with cetuximab would have resulted in a negative study. Determining the optimal radiation fractionation schedules to use with the various targeted agents may present an ongoing challenge. Sobering parallels may be drawn from the now nearly completed search for the ideal schedule to use with decades-old systemic agents. Results from the recently updated MACH-NC suggest that the survival benefit seen with concurrent chemotherapy is similar irrespective of the

11  Translational Research in Head and Neck Oncology

radiation fractionation regimen utilized (conventional HR 0.83 [95% CI 0.78–0.88] vs. altered HR 0.73 [95% CI 0.65– 0.82] p = 0.14) [64]. The results of RTOG 0129, which tests conventional versus accelerated fractionation, will help to determine the optimal radiation fractionation scheme to use with platinum-based chemotherapy. To re-emphasize the fact that more does not always mean better, a GORTEC phase III study showed no difference in progression-free survival at 3  years between accelerated versus conventional radiation therapy with concomitant carboplatin and 5-FU [68].

Where The location of the primary tumor site has been suggested to influence survival. A multivariate analysis of 492 patients showed better prognosis in patients treated for larynx and nasopharyngeal tumors compared to those with oropharynx, oral cavity, and hypopharyngeal primaries [69]. In another series of locally advanced patients treated with intra-arterial cisplatin and radiation (RADPLAT), those with hypopharyngeal primaries were more likely to develop distant metastases (odds ratio 2.8) compared to patients with oral cavity, oropharynx, or laryngeal tumors [70]. In the Bonner trial, 253 of the 424 patients in the study had oropharyngeal tumors. On subgroup analysis, these patients appeared to derive the greatest benefits in locoregional control and survival from the addition of cetuximab [29]. Whether or not this benefit reflects the influence of HPV-associated malignancy in the oropharynx is unknown. These findings further underscore the complexities facing the successful translation of targeted agents into clinical practice. Future prospective trials will likely need to focus on specific head and neck cancer subsites to avoid potential dilution of successful outcomes by the inclusion of possibly “non-responding” patients. In the case of oropharyngeal tumors, these will need to be further subdivided according to HPV status. At the same time, excessive stratification and selection of patients may severely cripple study power and applicability of results to the general head and neck cancer population.

Why The need to confirm hypotheses in prospective trials is highlighted by several pitfalls in the translation of the very logical and rational hypoxia story into clinical practice. Since 1912, when Swartz observed less severe skin reactions when a radiation source compressed the surrounding blood flow, careful clinical and laboratory research has subsequently

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established the significant role hypoxia plays in cancer progression and increased resistance to radiation and chemotherapy [71, 72]. In head and neck cancer, studies directly measuring pretreatment intratumoral oxygenation levels in primary tumors and lymph node metastases using polarographic electrode techniques predicted for response to radiation therapy [73] and was prognostic for disease-free survival [74]. More recent studies have focused on less invasive methods such as hypoxia-related biomarkers and functional imaging studies to correlate tumor hypoxia with treatment-related outcomes [75]. Using tissue samples from RTOG 90-03 patients, expression of lysyl oxidase, a hypoxia-related protein, was shown to be strongly associated with increased metastases, disease progression, and death [76]. This rationale lead to the testing of therapeutic strategies designed to ameliorate or target hypoxia. Anemia, which contributes to tumor hypoxia, is associated with inferior outcomes following both radiotherapy alone and concurrent chemoradiation [77–79]. However, correction of anemia has not improved treatment outcome in prospective trials. In one series of patients treated with sequential chemotherapy followed by chemoradiation, the use of blood transfusions to maintain hemoglobin levels >12 g/dL was associated with worse survival [80]. Two randomized DAHANCA studies that incorporated blood transfusions for low hemoglobin levels showed no benefit [81, 82]. Both erythropoietin [83] and darbepoietin alfa [84] reversed the effects of anemia on radiation response in preclinical models. Moreover, a retrospective study of patients treated with neoadjuvant chemoradiation and surgery for oral cavity/oropharyngeal cancers, the use of recombinant human erythropoietin completely abrogated the negative prognostic impact associated with hemoglobin levels <14.5  g/dL [85]. However, two randomized phase III trials showed no benefit to the addition of erythropoietin in anemic HNC patients undergoing radiation therapy [86, 87]. In fact, the Henke study resulted in poorer disease control and survival in patients randomized to receive erythropoietin [86]. A randomized study in cervix cancer patients was closed prematurely due to concern for increased thromboembolic events with erythropoietin [88]. A Cochrane review including 13,933 cancer patients in 53 trials showed that erythropoiesis-stimulating agents were associated with increases in on-study mortality and worse overall survival [89]. These unexpected clinical findings stimulated laboratory research that demonstrated expression of erythropoietin receptors on tumor cells in a variety of malignancies, including squamous cell carcinomas of the head and neck [90]. Potential erythropoietin-mediated signaling mechanisms responsible for increased cancer cell survival have been implicated [91, 92]. An alternate strategy of specifically targeting hypoxic cancer cells lead to the study of bioreductive agents such as tirapazamine [93]. Preclinical data showed preferential

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cytotoxicity to hypoxic tumor cells, and early phase I/II data demonstrated encouraging results when this agent was combined with chemotherapy and/or radiation [94–96]. However, two randomized phase III studies have shown no benefit from the addition of tirapazamine to radiation and chemotherapy. The HEADSTART trial showed no benefit in patients with locally advanced HNC treated to 70  Gy with three cycles of concurrent cisplatin [97]. The TRACE study, which used the same treatment scheme, was terminated early due to excessive mortality in the experimental tirapazamine arm [98]. Unfortunately, no systematic assessment of tumor hypoxia was performed in either of these trials. Studies using electrode and PET-based techniques suggest that approximately one third to one half of HNC patients do not have significant levels of tumor hypoxia [99, 100]. Therefore, it is possible that both of these trials were “biologically underpowered” to address the hypoxia question which was being investigated. Translational studies using functional imaging modalities that correlate with tumor hypoxia may better identify candidates for hypoxia-targeted therapy [101]. A substudy of TROG-98.02 using 18-F misonidazole-PET to image tumor hypoxia found a significantly higher risk of locoregional failure in hypoxic patients who received concurrent chemotherapy compared to those who also received tirapazamine [100]. The ability to image hypoxia-specific regions with PET and/ or functional MRI may further allow for physical targeting and treatment intensification with radiation techniques such as intensity-modulated radiation therapy [102]. However, significant daily fluctuations in tumor hypoxia imaging have been seen in as many as 30% of patients [103]. This suggests widespread clinical application will require further translational research into the dynamic nature of these processes studied by functional imaging modalities – vascular permeability, perfusion, and metabolism.

Conclusion Successful translational research will help to define new standards of care by improving the therapeutic ratio between treatment efficacy and toxicity. Better prognostic tools and more robust predictive assays will help to improve patient selection, stratifying patients to appropriate intensifications or deintensifications of therapy, and identifying those most likely to benefit from various treatments. In future trials, enriching the study population with those most likely to need and respond to certain therapies will hopefully magnify any potential improvements in outcome, in turn lowering the number of subjects needed to detect statistically significant differences. This is especially critical for head and neck cancer where the eligible patient pool from which to draw is smaller than other disease sites.

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New experimental therapies will need to be built on the foundation of prior successes, incorporating themselves into optimized standard of care regimens. Due to increasing economic constraints, leadership and guidance will likely need to come from the large umbrella cooperative groups such as the RTOG and EORTC regarding trial design and priorities. The design of trials should continue to combine treatment interventions with various correlative studies to identify and validate predictors that will help determine who benefits most from specific therapies. Strategic plans within RTOG have been discussed to improve the ability to perform more successful translational studies – tissue banking, seed grants, bioinformatics, and statistical support [104]. The war on cancer has seen decades of translational research create a new generation of targeted weapons with increasing specificity and accuracy. The danger now lies in using these agents to carpet-bomb entire patient populations, failing to commit the same level of resources to identifying the correct human targets.

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D.S. Yoo and D.M. Brizel from head and neck cancer. A multivariate study of 492 cases. Cancer. 1992;69(5):1224–34. 70. Doweck I, Robbins KT, Vieira F. Analysis of risk factors predictive of distant failure after targeted chemoradiation for advanced head and neck cancer. Arch Otolaryngol Head Neck Surg. 2001; 127(11):1315–8. 71. Bernier J, Hall EJ, Giaccia A. Radiation oncology: a century of achievements. Nat Rev Cancer. 2004;4(9):737–47. 72. Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev. 2007;26(2):225–39. 73. Nordsmark M, Overgaard M, Overgaard J. Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck. Radiother Oncol. 1996;41(1):31–9. 74. Brizel DM, Sibley GS, Prosnitz LR, Scher RL, Dewhirst MW. Tumor hypoxia adversely affects the prognosis of carcinoma of the head and neck. Int J Radiat Oncol Biol Phys. 1997;38(2):285–9. 75. Le QT, Kong C, Lavori PW, et al. Expression and prognostic significance of a panel of tissue hypoxia markers in head-and-neck squamous cell carcinomas. Int J Radiat Oncol Biol Phys. 2007;69(1):167–75. 76. Le QT, Harris J, Magliocco AM, et al. Validation of lysyl oxidase as a prognostic marker for metastasis and survival in head and neck squamous cell carcinoma: Radiation Therapy Oncology Group Trial 90-03. J Clin Oncol. 2009;27(26):4281–6. 77. Prosnitz RG, Yao B, Farrell CL, Clough R, Brizel DM. Pretreatment anemia is correlated with the reduced effectiveness of radiation and concurrent chemotherapy in advanced head and neck cancer. Int J Radiat Oncol Biol Phys. 2005;61(4):1087–95. 78. Fortin A, Wang CS, Vigneault E. Effect of pretreatment anemia on treatment outcome of concurrent radiochemotherapy in patients with head and neck cancer. Int J Radiat Oncol Biol Phys. 2008; 72(1):255–60. 79. Rades D, Stoehr M, Kazic N, et  al. Locally advanced stage IV squamous cell carcinoma of the head and neck: impact of pre-radiotherapy hemoglobin level and interruptions during radiotherapy. Int J Radiat Oncol Biol Phys. 2008;70(4):1108–14. 80. Bhide SA, Ahmed M, Rengarajan V, et al. Anemia during sequential induction chemotherapy and chemoradiation for head and neck cancer: the impact of blood transfusion on treatment outcome. Int J Radiat Oncol Biol Phys. 2009;73(2):391–8. 81. Overgaard J, Hansen HS, Overgaard M, et al. A randomized double-blind phase III study of nimorazole as a hypoxic radiosensitizer of primary radiotherapy in supraglottic larynx and pharynx carcinoma. Results of the Danish Head and Neck Cancer Study (DAHANCA) Protocol 5-85. Radiother Oncol. 1998;46(2): 135–46. 82. Overgaard J, Hansen HS, Specht L, et al. Five compared with six fractions per week of conventional radiotherapy of squamous-cell carcinoma of head and neck: DAHANCA 6 and 7 randomised controlled trial. Lancet. 2003;362(9388):933–40. 83. Stuben G, Pottgen C, Knuhmann K, et al. Erythropoietin restores the anemia-induced reduction in radiosensitivity of experimental human tumors in nude mice. Int J Radiat Oncol Biol Phys. 2003;55(5):1358–62. 84. Ning S, Hartley C, Molineux G, Knox SJ. Darbepoietin alfa potentiates the efficacy of radiation therapy in mice with corrected or uncorrected anemia. Cancer Res. 2005;65(1):284–90. 85. Glaser CM, Millesi W, Kornek GV, et  al. Impact of hemoglobin level and use of recombinant erythropoietin on efficacy of preoperative chemoradiation therapy for squamous cell carcinoma of the oral cavity and oropharynx. Int J Radiat Oncol Biol Phys. 2001;50(3):705–15. 86. Henke M, Laszig R, Rube C, et al. Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial. Lancet. 2003;362(9392):1255–60.

11  Translational Research in Head and Neck Oncology 87. Machtay M, Pajak TF, Suntharalingam M, et al. Radiotherapy with or without erythropoietin for anemic patients with head and neck cancer: a randomized trial of the Radiation Therapy Oncology Group (RTOG 99-03). Int J Radiat Oncol Biol Phys. 2007; 69(4):1008–17. 88. Thomas G, Ali S, Hoebers FJ, et al. Phase III trial to evaluate the efficacy of maintaining hemoglobin levels above 12.0 g/dL with erythropoietin vs above 10.0 g/dL without erythropoietin in anemic patients receiving concurrent radiation and cisplatin for cervical cancer. Gynecol Oncol. 2008;108(2):317–25. 89. Bohlius J, Schmidlin K, Brillant C, et al. Erythropoietin or Darbepoetin for patients with cancer – meta-analysis based on individual patient data. Cochrane Database Syst Rev. 2009;(3):CD007303. 90. Arcasoy MO, Amin K, Chou SC, Haroon ZA, Varia M, Raleigh JA. Erythropoietin and erythropoietin receptor expression in head and neck cancer: relationship to tumor hypoxia. Clin Cancer Res. 2005;11(1):20–7. 91. Pajonk F, Weil A, Sommer A, Suwinski R, Henke M. The erythropoietin-receptor pathway modulates survival of cancer cells. Oncogene. 2004;23(55):8987–91. 92. Winter SC, Shah KA, Campo L, et al. Relation of erythropoietin and erythropoietin receptor expression to hypoxia and anemia in head and neck squamous cell carcinoma. Clin Cancer Res. 2005;11(21):7614–20. 93. Zeman EM, Brown JM, Lemmon MJ, Hirst VK, Lee WW. SR-4233: a new bioreductive agent with high selective toxicity for hypoxic mammalian cells. Int J Radiat Oncol Biol Phys. 1986;12(7):1239–42. 94. Rischin D, Peters L, Hicks R, et  al. Phase I trial of concurrent tirapazamine, cisplatin, and radiotherapy in patients with advanced head and neck cancer. J Clin Oncol. 2001;19(2):535–42. 95. Le QT, Taira A, Budenz S, et al. Mature results from a randomized Phase II trial of cisplatin plus 5-fluorouracil and radiotherapy with or without tirapazamine in patients with resectable Stage IV head and neck squamous cell carcinomas. Cancer. 2006;106(9):1940–9.

189 96. Lunt SJ, Telfer BA, Fitzmaurice RJ, Stratford IJ, Williams KJ. Tirapazamine administered as a neoadjuvant to radiotherapy reduces metastatic dissemination. Clin Cancer Res. 2005;11(11): 4212–6. 97. Rischin D, Peters L, O'Sullivan B, et al. Tirapazamine, cisplatin, and radiation versus cisplatin and radiation for advanced squamous cell carcinoma of the head and neck (TROG 02.02, HeadSTART): a phase III trial of the Trans-Tasman Radiation Oncology Group. J Clin Oncol. 2010;28(18):2989–95. 98. Seiwert TY, Salama JK, Vokes EE. The chemoradiation paradigm in head and neck cancer. Nat Clin Pract Oncol. 2007;4(3): 156–71. 99. Brizel DM, Dodge RK, Clough RW, Dewhirst MW. Oxygenation of head and neck cancer: changes during radiotherapy and impact on treatment outcome. Radiother Oncol. 1999;53(2):113–7. 100. Rischin D, Hicks RJ, Fisher R, et  al. Prognostic significance of [18F]-misonidazole positron emission tomography-detected tumor hypoxia in patients with advanced head and neck cancer randomly assigned to chemoradiation with or without tirapazamine: a substudy of Trans-Tasman Radiation Oncology Group Study 98.02. J Clin Oncol. 2006;24(13):2098–104. 101. Le QT. Identifying and targeting hypoxia in head and neck cancer: a brief overview of current approaches. Int J Radiat Oncol Biol Phys. 2007;69(2 Suppl):S56–8. 102. Lee NY, Le QT. New developments in radiation therapy for head and neck cancer: intensity-modulated radiation therapy and hypoxia targeting. Semin Oncol. 2008;35(3):236–50. 103. Lin Z, Mechalakos J, Nehmeh S, et al. The influence of changes in tumor hypoxia on dose-painting treatment plans based on 18F-FMISO positron emission tomography. Int J Radiat Oncol Biol Phys. 2008;70(4):1219–28. 104. Chung CH, Wong S, Ang KK, et al. Strategic plans to promote head and neck cancer translational research within the radiation therapy oncology group: a report from the translational research program. Int J Radiat Oncol Biol Phys. 2007;69(2 Suppl):S67–78.

Chapter 12

Preclinical Models of Head and Neck Squamous Cell Carcinoma Michiel W.M. van den Brekel, C.L. Zuur, Stephen P. Malkoski, and Xiao-Jing Wang

Abstract  Model systems are irreplaceable to study cancer. Although the best model system is human cancer in man, research in patients is restricted by ethical and financial restraints. Furthermore, experiments cannot be repeated and patient numbers are limited. Tumor cell cultures are the most versatile system to study cancer cells. They allow for repeated experiments in controlled conditions, are relatively inexpensive and are ideal to study genes, pathways, and tumor response. Mouse models enable to study cancer behavior and carcinogenesis in vivo and many different model systems are available. Apart from xenografts in immune compromised mice, transplantation of oral mice tumors in syngeneic mice, animals developing oral cancer using carcinogen exposure and genetically modified mice can be used. All these models, however, have advantages and limitations that will be discussed in this chapter. Keywords  Squamous cell cancer • Mouse • Model system • Xenograft • Cell culture • Head and neck • Transgenic

Head and neck squamous cell carcinomas (HNSCCs) represent 3–5% of all newly diagnosed cancers each year in the western world with 5-year survival rates in the order of 25–95% depending on disease site and stage. The limited survival rates in most patients indicate the need for novel treatment strategies with new potent drugs. In addition, these survival rates yield a widely divergent individual response of similar histopathological cancers to the applied treatment regimen. Currently, the decision on therapy relies mainly on the outcome of both retrospective data as well as various well-performed prospective trials and meta-analyses. However, so far no prospective trial has been conducted using biomarkers for treatment selection and thus we have

M.W.M. van den Brekel (*) Otorhinolaryngology – Head and Neck Surgery Department, Netherlands Cancer Institute – Antoni van Leeuwenhoek Hospital and Academic Medical Center, University of Amsterdam, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands e-mail: [email protected]

not been able to stratify patients based on individual tumor properties since knowledge regarding the biological basis of variations in tumor response to chemotherapeutics was and is still limited. HNSCCs are characterized by a rather large genetic diversity, possibly caused by the long duration of carcinogenic exposure and the genetic instability of most head and neck carcinomas [1]. However, several pathways are almost always invariably involved in carcinogenesis, such as the P53 and INK4a pathways [2, 3]. The fact that these tumors are genetically highly heterogeneous and instable has hampered the development of drugs specifically targeting pathways relevant in head and neck cancer. So far, only the inhibition of the EGFR receptor has proven to have clinical benefit for a subpopulation of head and neck cancer patients, especially when combined to radiotherapy [4]. However, in preclinical models, several other targeted therapies have shown promising results, such as drugs targeting phosphoinositol (PI)-3-kinase–AKT, insulin-like growth receptor, BCL2, MET, and several others [5–8]. Although the role of human papilloma virus (HPV) was postulated a long time ago, only recently it was recognized that HNSCCs can be divided really into those that are and those that are not associated with HPV [9]. The HPV16 papillomavirus oncogenes E6 and E7 have been detected in HPV genomes in HNSCC and its oncoproteins are known for their ability to bind and inactivate tumor suppressor proteins p53 and retinoblastoma (pRb) [10]. Tumors with HPV infection occur at a younger age, are less related to smoking and alcohol and do not have P53 mutations or loss of P16 (INK4a) function by mutations, deletions, or methylation. Instead, these pathways are deregulated by the E4 and E6 proteins expressed by the virus [11]. HPV status does not only predict treatment outcome, but likely, should also be used to guide treatment [12]. However, as stated above, at the moment we lack the knowledge and reliable trials to personalize treatment regimens in HNSCC. Studying cancer in humans poses enormous ethical, financial, and practical hurdles, due to the limited number of patients and tumor material, the enormous costs, and the ethical dilemmas in clinical research. Therefore, preclinical

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models are an important tool for exploring tumor initiation and progression, cancer genetics, and novel therapeutic approaches. A variety of HNSCC model systems have been developed, including cancer cell lines derived from human HNSCC, exposure of animals to oral carcinogens, and genetically engineered mouse models (GEMMs). Each system has important strengths and weaknesses that must be appreciated to interpret data derived from these models. To maximize clinical relevance, model systems should resemble human HNSCC as closely as possible. For example, cell lines should harbor the genetic and epigenetic alterations common to HNSCC and carcinogen exposures should mimic the routes and chemicals associated with human HNSCC. Similarly, GEMMs should examine the genetic alterations frequently observed in human HNSCC. To overcome the intrinsic limitations of a given model, results should be validated by multiple approaches in different systems; however, ultimately, all results obtained in model systems must be validated in human samples or subjects.

HNSCC Cell Lines Cancer cell lines are the most versatile model system wherein cancer cells can be characterized and even manipulated genetically. Genetic manipulation techniques have enabled us to study the influence of specific genetic abnormalities or correction of these abnormalities on tumorigenesis, tumor behavior, or treatment response [5]. Using these techniques, one can study human genes in mouse cell lines as, e.g., the influence of known mutagens on human P53 [13]. In addition, it has been shown that differences in response can be attributed to differences in the genetic make-up of HNSCC cell lines, them being either HPV positive or negative [8] and Li et  al. showed that by blocking SRC kinase, cetuximab resistant tumors can become sensitive again [14]. Such experiments, that can only be done using well-characterized cancers, can give us insights that in the near future may lead to more individualized and more effective treatment protocols. Although in HNSCC the routine use of molecular markers for treatment selection is not established yet, in several other tumor types such as breast, colon, and lung, this molecular knowledge has already been translated into important predictive assays used in treatment selection [15, 16]. Currently, there is a strong urge to find, validate, and implement markers for a more individualized treatment selection in HNSCC. A major advantage of cell lines is that experiments can be done within several days and are relatively inexpensive. Using cell lines, the same tumors can be tested over and over again with multiple new drugs, combinations of drugs, or genetic interventions. This enables testing numerous

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radiotherapy doses or drugs on the same tumor as well as the mechanisms or conditions by which tumors become resistant to treatment. These mechanisms can then be targeted to avoid resistance. For instance, in human HNSCCs, ligand activation of the epidermal growth factor receptor (EGFR) leads to downstream signaling of several prosurvival cascades (pathways) eventually promoting cell proliferation, angiogenesis, invasion, and metastasis. Therefore, EGFR is one of the most promising molecular targets in cancer therapy. Recently, Li et al. described several mechanisms of acquired resistance of SCC to cetuximab (an EGFR-blocking antibody) and performed research to investigate the role of nEGFR (EGFR translocated from plasma membrane to nucleus) in this phenomenon using nonsmall cell lung carcinoma cell lines. In these cells, an increased Src family kinase (SFK) activity was found, linked to the translocation of the EGFR to the nucleus, suggesting that a combined modality treatment regimen of blocking SFKs together with cetuximab may be a future clinical trial treatment design for patients with EGFR resistant tumors [14]. In the field of studying radiotherapy and radiosensitization in head and neck cancer, much work has been done using cell lines [17]. It has been shown that hypoxia, DNA repair, and repopulation, as well as intrinsic tumor cell characteristics play an important role in radioresistance [18, 19]. The role of HPV has also been studied in head and neck cell lines. Mouse tonsil epithelial cell lines (MTECs) become immortalized by HPV 16 E6–E7 transfection and allow for extensive research to determine what viral genes are required for this immortalization and anchorage-independent growth and, eventually, malignant growth in vivo. Hoover et al. in 2007 reported that HPV viral oncogenes alone were indeed sufficient to induce anchorage-independent growth of MTECs in vitro, but additional H-ras oncogene expression was needed to form invasive cancers in vivo [20]. However, cancer cell lines also have critical limitations. Most importantly, they represent a homogeneous clonal population capable of growing in vitro; in fact, the majority of individual tumors and cancer cells within an individual tumor are incapable of growing in tissue culture. Hence, cultured cells typically fail to reflect the genetic heterogeneity of the native tumor from which they were derived. Interestingly, patients whose tumors can establish cell lines have worse clinical prognosis [21], suggesting that characteristics supporting in  vitro growth are indicative of aggressive tumor behavior in vivo. Furthermore, as cells are passaged, there is increased selective pressure for in  vitro growth and after many passages, cultured cancer cells may differ from the original tumor from which they were derived. For example, tumor lines and native tumors may exhibit different chemosensitivity patterns and this can be influenced by the number of in  vitro passages [22–24]. Culture conditions can also influence the responses to cytotoxic therapies; e.g., cells

12  Preclinical Models of Head and Neck Squamous Cell Carcinoma

grown as anchorage-independent spheroids can have different responses to cytotoxic agents than the same cells grown as anchorage-dependent monolayers [25]. Accordingly, cell lines cannot be used to predict treatment response in individual patients [26]. Many of these issues have potentially been accentuated in HNSCC as there is a relative paucity of well characterized lines [21, 27], and a lack of standardized tissue culture techniques that can limit reproducibility [28–34]. A final important limitation of cultured cells is an inability to study the interactions between tumor epithelial cells and key components of the tumor stroma, including fibroblasts, immune cells, and the vasculature. Despite these limitations, much of our basic mechanistic understanding of the roles of specific molecules has been derived from cell culture data. Perhaps the best successful example of basic biologic understanding directly improving cancer outcome occurred in chronic myelogenous leukemia (CML) where the observation that inhibition of the bcr–abl fusion protein reduced growth of leukemic cell lines led to the successful clinical deployment of imatinib [35, 36]. Unfortunately, because HNSCC appears to be more genetically heterogeneous than CML, HNSCC may not be susceptible to inhibition of a single oncogenic pathway [1]. It is probable that using a combination of drugs, inhibiting several pathways, such as EGFR inhibitors in combination with, e.g., blockade of the PI-3-kinase–AKT pathway, insulin-like growth factor receptor (IGFR), BCL2, or cMET holds promise for the future and has been studied in preclinical models [5–8]. Although cell lines are the optimal system to study pathways and the role of specific genes for carcinogenesis and treatment response, it has proven very difficult to find reliable markers from cell line experiments. We recently studied radioresistance in cell lines obtained from Grenman in Turku (Finland) and made a gene expression profile correlating with radiosensitivity [19, 37, 38]. Unfortunately, this expression profile was not predictive of local control after radiotherapy of laryngeal cancer in patients. This again shows the difficulty of extrapolating in vitro findings to clinical practice.

Short Term Cultures As cell lines are difficult to establish, as they represent only a fraction of a tumor and as in every passage additional genetic changes occur, cell lines cannot be used to guide treatment of an individual patient. To augment individual treatment planning, short-term culturing techniques are used. In this technique, a small tissue biopsy sample that includes both tumor epithelial cells and tumor stromal cells (e.g., fibroblasts) is cultured and then tested for sensitivity to chemotherapeutic agents in vitro. In this setting, it appears that a single biopsy (100  mg) sample is representative of the

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Fig. 12.1  Digital immunofluoroscopy of short-term cultured cells from a oropharyngeal carcinoma A tumor cell and fibroblast are shown (64×). Staining: cell nucleus: Hoechst staining double-stranded DNA (blue); cell actin: Alexa Fluor® 568 phalloidin (red); SCC cytokeratin: Mouse anti-human pan Cytokeratin and Alexa Fluor® anti-mouse IgG 568 (green)

entire tumor with respect to chemoresponsiveness [39] and that coculture of tumor and stromal cells increases the predictive value of this assay [40] (Fig. 12.1). One technical difficulty of this approach is the overgrowth of the fibroblast subpopulation; however, this can be overcome by avoiding enzymatic digestion and allowing both tumor epithelial and stromal cells to grow out of multicellular tumor particles. Short-term cultures can also be grown on a collagen spongegel-supported matrix to maintain tissue architecture and facilitate cell–cell interactions that may be important in chemotherapy response. Using these systems, a culture sufficient for in vitro drug testing can be established 80% of the time [41, 42] and in vitro testing can occur within a few days. In addition, short-term cultures can be subsequently used to establish xenograft models if desired (see below) [43]. In these experiments, the typical read out is cell count or proliferation after treatment with a cytotoxic agent [40, 44, 45], however, qualitative data, such as cell cycle arrest, differentiation, and morphology can also be collected to assess the specific response of the tumor and stroma cells to the (cancer) drug applied. For mass screening the HTS immunofluorescent automated microscopy with high-content imaging is possible using the CellProfiler program [46]. The tumor clonogenic assay is one of the most intensely studied in vitro methods for chemosensitivity testing and evaluates colony formation of cancer (stem) cells with the potential for anchorage-independent growth in semisolid media in which individual cells develop into colonies. Comparing response to drugs in this clonogenic assay and in patients, Fiebig et al. found that 62% of the comparisons for drug sensitivity and 92% of the comparisons for drug resistance were

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correct [41]. In further experiments in head and neck cancer using the histoculture drug-response assay, the correlation between clinical response to induction chemotherapy and the prediction in the assay was almost 78% [45]. An alternative method for short-term cultures is a collagen sponge-gel-supported histoculture in which tissue architecture is maintained. The architecture allows more cell–cell interaction which might be important in chemotherapy response. A histoculture drug response assay for individualizing chemotherapy has been developed and proved to be very predictive. As an end-point, the MTT assay can be used [40, 44, 45]. Comparison between all these culture methods has not been performed. Although small patient numbers were used, primary tumor cell culture models have been shown to predict the individual tumor sensitivity for different cancer drugs [45, 47–49]. However, there are so far no phase III studies demonstrating a significant increase in survival rates compared to empirically determined standard chemotherapy regimens. Therefore, the tissue culture has not yet found a routine role in the individualization of patient therapy.

Xenograft Mouse Models Another approach for amplifying, studying, and testing tumors in vivo is to subcutaneously implant human cancers into immunologically compromised nude or SCID mice. Depending on the original tumor subsite (oral cavity, oropharynx, larynx, and hypopharynx), 70–80% of the patient HNSCCs were successfully xenografted and short-term cultured in the mouse [43]. Once established, the system allows for in  vivo testing of novel cancer drugs, as studying the response of human tumors in subcutaneous, e.g., ectopic tissue sites of the mouse to the various cancer drugs applied may produce relevant and predictive information to the clinic, provided that pharmacokinetic parameters (especially dosing) are employed. Alternatively, orthotopic transplantation is suggested to facilitate metastatic spread thereby increasing the models’ clinical predictive value as various drugs can then be tested on either (or both) the primary tumor growing in a physiologically relevant site and distant metastatic disease, especially in case therapy is initiated at the point when metastases are macroscopic in nature [50]. To study the changes in gene expression with transformation and metastatic tumor progression of squamous cell carcinomas, oral tumors and cell lines derived from mice were transplanted into inbred syngeneic recipients [51]. Other examples include the oral SCC VII/SF cell line (from C3H/ HeJ mice) and the transformed PAM 212 cell line (from BALB/c keratinocytes) [52] and similar models have been described using a hamster buccal pouch carcinoma of rat oral

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carcinoma [53, 54]. In addition, tumor cell lines can be manipulated ex vivo then transplanted to study the roles of specific molecules or pathways during tumor progression and metastases. For example, while induction of HPV genes E6 and E7 can immortalize mouse tonsil epithelial cells in vitro, additional H-ras transduction is necessary to form invasive cancers [20]. Compared to cell lines, direct xenograft mouse models preserve key features that cells in culture derived from the same tumor samples irreversibly lose [55], perhaps by preserving the human stroma and immune cells important for tumor growth and metastases [56]. Xenografts derived directly from patient biopsies, with minimal in  vitro manipulation, appear to retain better the morphological and molecular marker of the source tumors, despite serial passing across several generations of mice [57]. In addition, human tumors can be serially transplanted into other immunocompromised mice providing additional tumor material for downstream molecular or cellular analysis or additional tumor-bearing mice for in  vivo testing of therapeutic compounds. These systems may be better suited for studying invasiveness and metastases than cell culture systems [58–60], particularly if coupled with evolving imaging techniques such as micro PET-CT. However, because the xenograft model involves implanting human tumors cells, it cannot be used to study early stage carcinogenesis, tumor initiation, or chemoprevention. Also, when tumors are transplanted they will still require angiogenesis and supporting tumor stroma from the murine host, and as recipient mice are immunocompromised, this model is not suitable for evaluating tumor immunology. Moreover, agent metabolism and pharmacodynamics are different in mice, and, as with immortalized cell lines, serial passaging of tumor xenografts can change the tumor characteristics by selecting for tumor cell populations suited to growing in an immunocompromised host [61, 62]. Finally, compared to experiments with cell lines, xenografting experiments are more time-consuming and expensive.

Cancer Induction by Chemical Carcinogens Mice, rats, and hamsters can be exposed to carcinogens to induce cancer. Although exposures can be laborious and timeconsuming, these models are especially useful to study carcinogenesis and chemopreventive strategies as there is usually a long latency between exposure and tumor development and animals frequently develop premalignant lesions [63]. Depending on the mutagen, exposure route, and dose, oral tumors with different genetic alterations and behaviors can be produced. Like human HNSCC, chemically induced HNSCC harbor a variety of genetic lesions, however, chemically

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induced tumors are typically more homogenous than their human counterparts as animals are only exposed to one carcinogen that produces characteristic genetic alterations as opposed to being exposed to a complex mixture of compounds each with different genotoxic effects (i.e., cigarette smoke) [64, 65]. Finally by applying carcinogenesis protocols to genetically engineered mice, the specific roles of molecules and pathways in promoting or inhibiting tumor initiation, growth, or metastases can be assessed. One well-characterized HNSCC model is application of the H-ras mutagen 7,12-dimethylbenz(a)anthracene (DMBA) to the hamster buccal pouch [66, 67]. In this model, oral DMBA is applied three times weekly for 10–24  weeks. Squamous cell carcinomas will occur in the majority of the hamsters, and lymph node metastases are sometimes found [68]. Because these tumors are almost all H-ras initiated, they do not typically have the genetic instability seen in human HNSCC where chromosome breaks and aneuploidy are frequent. Nonetheless, this is a clinically relevant genetic alteration as HNSCC arising in Asian patients is frequently initiated by activated Ras signaling [69]. One downside of this model is that the tools and reagents for hamsters are more limited than those for mice, however, DMBA can also be used to induce skin SCC in mice (when combined with tumor promotion by a phorbol ester) or to induce oral SCC in genetically susceptible animals [70]. Although DMBA is not a tobacco carcinogen, it is a convenient way of introducing H-ras mutations to the oral epithelium to evaluate the interactions of other experimental systems on Ras-initiated tumors. In a study of Chang et  al. in 2000, N-methyl-Nbenzylnitrosamine (MBN) was applied to hamster buccal pouches to characterize the MBN-induced tumors with regard to the frequency of p53 and H-ras mutations, as these are among the specific molecular alterations observed in human HNSCCs [71]. In this analysis, the alterations in p53, H-ras, and telomerase activity observed in the model are similar in many respects to the analogous human lesions of the head and neck, suggesting that this model system may be particularly useful for the development of cancer chemoprevention regimens and cancer therapies. Rats and mice also develop oral squamous cell carcinomas after application of the chemical carcinogen 4-nitroquinoline N-oxide (4-NQO) for 2–6 months in their drinking water or application in a concentrated solution to the oral cavity for 12–16 weeks [72]. 4-NQO, although not a natural tobacco derivative, causes a spectrum of DNA damage similar to that caused by tobacco-associated carcinogens. In addition, in p53 transgenic mice, the incidence of oral cancer was increased from 0 to 67% when treated with 4-NQO thrice weekly for 16 weeks and a maximum follow-up of 32 weeks [73]. Also in HPV16-transgenic mice treated with 4-NQO, the incidence of SCC was increased significantly compared

to their nontransgenic counterparts and histopathological analyses demonstrated progressive neoplastic disease in the oral cavity with remarkable similarities to human HPVpositive HNSCC. Using this model, the investigators reported to have identified a biomarker that distinguishes between HPV-positive and HPV-negative HNSCC [74]. The 4-NQO-induced cancer model has also been used to induce salivary gland cancer [75]. Furthermore, other carcinogens can be used, such as benzo[a]pyrene (B[a]P), N-nitroso-N-methylurea (NMU), 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone (NNK), or nitrosonornicotine (NNN). The oral cavity of hamsters, rats, and mice or the skin of these animals can be used to induce cancer [65, 76, 77]. As these animals first develop premalignant lesions, these models are especially useful to study inhibitory (chemopreventive) or promoting stimuli. It has been shown that DMBAinduced carcinogenesis in the hamster cheek pouch can be counteracted by long-term (18 weeks) topical application of GW2974, a dual inhibitor of EGFR and ErbB2 tyrosine kinase, decreasing the incidence, number, and size of both visible tumors and microscopic lesions such as hyperplasia, dysplasia, and SCC significantly [78]. In another study, celecoxib (a highly selective inhibitor of cyclooxygenase (COX)-2, known to be overexpressed in human (pre)malignant oral lesions) was applied for 7 weeks in the oral cavity of hamsters after they were painted for 5  weeks with DMBA. Celecoxib was effective in delaying the onset of early lesions and able to slow down the growth of the oral tumor [79]. The antilipidperoxidative and antioxidant potential of curcumin and piperine were reported to be crucial in the biochemical mechanistic pathway of their chemoprevention in DMBAinduced oral carcinogenesis [80, 81]. A major improvement of using carcinogen induced cancer has been made by using these carcinogens in genetically predisposed mice of cell lines, such as the P53 knock-out mice, or the HPV 16 E–E7 transfected immortalized oral cell lines [73, 74, 82]. Using carcinogens in these models causes cancer or transformation in a much faster and controlled way [83]. Using this model system, one can study the role of different genes on carcinogenesis. Apart from P53, also Xeroderma pigmentosa A (XPA) knock-out or cycline D1 overexpression have been shown to increase and accelerate oral cancer formation in 4-NQO-treated mice [84, 85].

Transgenic Mouse Models GEMMs have been an enormous step forward for cancer modeling and allow evaluation of discrete genetic alterations in specific organs in  vivo in an immunocompetent animal. Additional benefits of GEMMs include the ability to evaluate how multiple genetic defects interact to promote or inhibit

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cancer and the opportunity to evaluate whether specific targeted therapies are active against tumors with a defined genetic composition. Drawbacks are that human cancers are more genetically complex and heterogeneous than tumors produced in mouse models and differences in the human and mouse immune systems may complicate studies of tumor immunology. Advances in murine embryology and genetics initially facilitated targeted mutagenesis of the mouse germ line by homologous recombination in ES cells leading to the creation of classic “knock out” mice. If a genetic modification is not lethal during embryonic development, heterozygotes can be crossed to create mice homozygous for a particular gene deletion. While knockout mice can occasionally be used to study deletion of tumor suppressors, there are critical limitations to this approach. First, global gene deletion of putative tumor suppressors is frequently embryonic lethal and this prevents the assessment of many genes using this strategy. For similar reasons, it is difficult, if not impossible, to study combinations of genetic modifications using this technique [86]. In addition, because the genetic modification is present in all tissues, tumors can develop in multiple anatomic locations, potentially hindering study of the tumor of interest. Finally, the fact that tumor stromal cells (fibroblasts, immune cells, and vasculature) also harbor the same genetic modification can impact overall tumor behavior in unanticipated ways. So far, germ-line deletions have not provided HNSCC specific insight. A step toward conditional mutagenesis is to place oncogenes under control of a tissue-specific promoter. Examples are K14-HPV16 mice that express E6/E7 in K14 expressing cells. These mice develop hyperplasia and some strains also oral SCCs [87]. Recently, also AKT activation or Ras activation in combination with loss of P53 has been shown to induce oral cancer [88, 89]. With the development of conditional genetic manipulation systems, many of these problems have been overcome [90]. In these systems, a target gene is flanked by loxP restriction sites that are the target of the Escherichia coli bacteriophage P1 Cre recombinase; Cre recombinase then excises sequences between loxP sites, allowing conditional gene deletion. As animals harboring conditional alleles are phenotypically normal in the absence of genetic recombination mediated by Cre recombinase, this system avoids many of the problems of embryonic lethality or infertility associated with germ-line deletions. By placing a loxP-flanked stop codon upstream of an oncogene (e.g., KrasG12D), this approach can also be used to “knock-in” tumor initiators [91, 92] or specific p53 mutations. Tissue restricted genetic manipulation is achieved by delivery of Cre recombinase to the cells of interest. While this can be done with adenoviral vectors [91], this approach has largely given way to transgenic approaches that use a tissue-specific promoter to target Cre recombinase expression

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to the cells of interest [93]. In this setting, genetic manipulation then occurs only in cells that express the targeted Cre recombinase transgene. The Epstein–Barr virus ED-L2 promoter as well as keratin 5 (K5) and keratin 14 (K14) promoters have been used to target gene manipulations to the oral epithelium [94–98], however, because keratins are robustly expressed in a variety of epithelial tissues, especially the skin and mammary tissue, an additional layer of control is required to restrict Cre recombinase expression to the head and neck epithelium. This is achieved by using a ligand-inducible Cre recombinase fusion protein whose expression is restricted by a K5 or K14 promoter. Currently available constructs include both tamoxifen-inducible truncated estrogen receptor fusions, such as K14CreERT and K5CreERT2 [93, 95] (Figs.  12.2 and 12.3); and RU486-inducible truncated progesterone receptor fusions, such as K14CrePR or K5CrePR [99], although only the CrePR constructs have so far been used to generate mouse models of HNSCC [96]. Another system in which genes can be turned on and off is the tetracycline-inducible system (tet-on and tet-off receptor) targeted to epithelial cells combined with oncogene under the control of tet-regulated responsive elements. On doxycycline administration the oncogene can be expressed [100, 101]. The main advantage of ligand-dependent systems is that they allow tissue-specific, spatial, and temporal control of recombination. Because these systems can be used to introduce

Fig.  12.2  Coronal histopathological section through the mouth and nose of a K14P53FF transgenic mouse (nonfunctioning P53 in all K14 expressing cells). Some of these animals develop oral squamous cancer as visible on the right side around the mandible

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either ionizing radiation or poly(ADP-ribose) polymerase (PARP) inhibitor-induced cell death. Given that Smad4 expression is frequently reduced in HNSCC this may have substantial clinical implications [96]. There is a great need to develop more reliable and different GEMMs for HNSCCs. When these are established, they should be validated for predicting treatment responses in human HNSCCs. Also the influence of different genetic make-up on tumor behavior and treatment response is an important aspect to be studied. So far HNSCC model systems are not as well developed as breast cancer models and pulmonary cancer models. It is a challenge for the next years to catch up with these research fields.

References

Fig. 12.3  Transgenic mouse with K14P53FF and P16 knockout with a cheek cancer

multiple somatic genetic alterations simultaneously into a target tissue interactions between different oncogenes and tumor suppressors can be evaluated in vivo. Disadvantages of this system are that most inducible Cre recombinase systems have some level of background activity and toxicity, and that there may be variability in recombination efficiency for different genes, partially related to the distance between LoxP sites [102, 103]. Apart from using tissue-specific promoters and ligands, an alternative is to use an adenoviral vector: adenoCre. This system is used in pulmonary cancer [91]. GEMMs can be used to test whether alterations in specific pathways or combinations of pathways are sufficient for HNSCC development and the mechanisms by which specific molecular alterations contribute to HNSCC development [96, 98, 104]. For example, although knock-in of oncogenic KrasG12D in the oral cavity causes benign papilloma formation [97], simultaneous deletion of transforming growth factor beta type II receptor (TGFbetaRII) with KrasG12D activation causes full penetrance HNSCC [70]. Thus it appears that Kras activation functions as a tumor initiator while defective TGFbeta signaling causes tumor progression, especially as TGFbetaRII deletion in the oral epithelium does not cause HSNCC. Interestingly, in contrast to TGFbetaRII, Smad deletion in the oral epithelium causes spontaneous HNSCC, suggesting that although both these molecules are components of the TGFbeta signaling pathway that they have distinct nonoverlapping functions in HNSCC [96, 104]. GEMMs can also be used to suggest novel therapeutic avenues. For example, HNSCC induced by Smad4 deletion have increased genetic instability and may hence be more susceptible to

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71. Chang KW, Sarraj S, Lin SC, Tsai PI, Solt D. P53 expression, p53 and Ha-ras mutation and telomerase activation during nitrosaminemediated hamster pouch carcinogenesis. Carcinogenesis. 2000;21:1441–51. 72. Schoop RA, Noteborn MH, de Jong Baatenburg RJ. A mouse model for oral squamous cell carcinoma. J Mol Histol. 2009;40: 177–81. 73. Zhang Z, Wang Y, Yao R, Li J, Lubet RA, You M. p53 transgenic mice are highly susceptible to 4-nitroquinoline-1-oxide-induced oral cancer. Mol Cancer Res. 2006;4:401–10. 74. Strati K, Pitot HC, Lambert PF. Identification of biomarkers that distinguish human papillomavirus (HPV)-positive versus HPVnegative head and neck cancers in a mouse model. Proc Natl Acad Sci USA. 2006;103:14152–7. 75. Ide F, Kitada M, Sakashita H, Kusama K. Reduction of p53 dosage renders mice hypersensitive to 7, 12-dimethylbenz(alpha) anthracene-induced salivary gland tumorigenesis. Anticancer Res. 2003;23:201–4. 76. Yuan B, Heniford BW, Ackermann DM, Hawkins BL, Hendler FJ. Harvey ras (H-ras) point mutations are induced by 4-nitroquinoline-1-oxide in murine oral squamous epithelia, while squamous cell carcinomas and loss of heterozygosity occur without additional exposure. Cancer Res. 1994;54:5310–7. 77. Tang XH, Knudsen B, Bemis D, Tickoo S, Gudas LJ. Oral cavity and esophageal carcinogenesis modeled in carcinogen-treated mice. Clin Cancer Res. 2004;10:301–13. 78. Sun Z, Sood S, Li N, et  al. Chemoprevention of 7, 12-dimethy­ lbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamster cheek pouch by topical application of a dual inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 tyrosine kinases. Oral Oncol. 2008;44:652–7. 79. Feng L, Wang Z. Chemopreventive effect of celecoxib in oral precancers and cancers. Laryngoscope. 2006;116:1842–5. 80. Manoharan S, Balakrishnan S, Menon VP, Alias LM, Reena AR. Chemopreventive efficacy of curcumin and piperine during 7, 12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. Singapore Med J. 2009;50:139–46. 81. Letchoumy PV, Mohan KV, Stegeman JJ, Gelboin HV, Hara Y, Nagini S. In vitro antioxidative potential of lactoferrin and black tea polyphenols and protective effects in vivo on carcinogen activation, DNA damage, proliferation, invasion, and angiogenesis during experimental oral carcinogenesis. Oncol Res. 2008;17: 193–203. 82. Zhong LP, Pan HY, Zhou XJ, et al. Characteristics of a cancerous cell line, HIOEC-B(a)P-96, induced by benzo(a)pyrene from human immortalized oral epithelial cell line. Arch Oral Biol. 2008;53:443–52. 83. Attardi LD, Donehower LA. Probing p53 biological functions through the use of genetically engineered mouse models. Mutat Res. 2005;576:4–21. 84. Ide F, Kitada M, Sakashita H, Kusama K, Tanaka K, Ishikawa T. p53 haploinsufficiency profoundly accelerates the onset of tongue tumors in mice lacking the xeroderma pigmentosum group A gene. Am J Pathol. 2003;163:1729–33. 85. Wilkey JF, Buchberger G, Saucier K, et al. Cyclin D1 overexpression increases susceptibility to 4-nitroquinoline-1-oxide-induced dysplasia and neoplasia in murine squamous oral epithelium. Mol Carcinog. 2009;48:853–61. 86. Berns A. Cancer. Improved mouse models. Nature. 2001;410: 1043–4. 87. Coussens LM, Hanahan D, Arbeit JM. Genetic predisposition and parameters of malignant progression in K14-HPV16 transgenic mice. Am J Pathol. 1996;149:1899–917. 88. Moral M, Segrelles C, Lara MF, et al. Akt activation synergizes with Trp53 loss in oral epithelium to produce a novel mouse model for head and neck squamous cell carcinoma. Cancer Res. 2009;69:1099–108.

200 89. Raimondi AR, Molinolo A, Gutkind JS. Rapamycin prevents early onset of tumorigenesis in an oral-specific K-ras and p53 two-hit carcinogenesis model. Cancer Res. 2009;69:4159–66. 90. Akagi K, Sandig V, Vooijs M, et  al. Cre-mediated somatic sitespecific recombination in mice. Nucleic Acids Res. 1997;25: 1766–73. 91. Meuwissen R, Linn SC, van der Valk M, Mooi WJ, Berns A. Mouse model for lung tumorigenesis through Cre/lox controlled sporadic activation of the K-Ras oncogene. Oncogene. 2001;20: 6551–8. 92. Chan IT, Kutok JL, Williams IR, et al. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J Clin Invest. 2004;113:528–38. 93. Bindels EM, van den Brekel MW. Development of a conditional mouse model for head and neck squamous cell carcinoma. Adv Otorhinolaryngol. 2005;62:1–11. 94. Opitz OG, Harada H, Suliman Y, et al. A mouse model of human oral-esophageal cancer. J Clin Invest. 2002;110:761–9. 95. Vasioukhin V, Degenstein L, Wise B, Fuchs E. The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc Natl Acad Sci USA. 1999;96:8551–6. 96. Bornstein S, White R, Malkoski S, et al. Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation. J Clin Invest. 2009;119:3408–19.

M.W.M. van den Brekel et al. 97. Caulin C, Nguyen T, Longley MA, Zhou Z, Wang XJ, Roop DR. Inducible activation of oncogenic K-ras results in tumor formation in the oral cavity. Cancer Res. 2004;64:5054–8. 98. Lu SL, Herrington H, Wang XJ. Mouse models for human head and neck squamous cell carcinomas. Head Neck. 2006;28:945–54. 99. Matsumoto T, Kiguchi K, Jiang J, et al. Development of transgenic mice that inducibly express an active form of c-Src in the epidermis. Mol Carcinog. 2004;40:189–200. 100. Vitale-Cross L, Amornphimoltham P, Fisher G, Molinolo AA, Gutkind JS. Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis. Cancer Res. 2004;64:8804–7. 101. Paillard F. “Tet-on”: a gene switch for the exogenous regulation of transgene expression. Hum Gene Ther. 1998;9:983–5. 102. Higashi AY, Ikawa T, Muramatsu M, et al. Direct hematological toxicity and illegitimate chromosomal recombination caused by the systemic activation of CreERT2. J Immunol. 2009;182: 5633–40. 103. Leonhard WN, Roelfsema JH, Lantinga-van Leeuwen IS, Breuning MH, Peters DJ. Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice. BMC Biotechnol. 2008;8:18. 104. Opitz OG, Quante M, von Werder A, Heeg S, Blum HE. A mouse model of oral-esophageal carcinogenesis. Onkologie. 2005;28: 44–8.

Chapter 13

Morphologic Investigations in Head and Neck Cancer Suyash Mohan and Gaurang V. Shah

Abstract  Head and neck cancer (HNC) is the fifth most common cancer worldwide and is traditionally associated with high morbidity and mortality. Patients with head and neck squamous cell cancer require a careful evaluation and a multidisciplinary team approach to determine optimal management. Treatment planning depends on TNM staging, which is evaluated with physical examination, endoscopies, and crosssectional imaging. CT and MR imaging form the main stays of cross-sectional imaging and is extensively utilized in characterizing and staging of malignant tumors involving the head and neck which is critical for the selection of appropriate therapeutic regimens. The goal of imaging in patients with HNC are to establish tumor extent and size, to assess nodal disease, for possible perineural tumor spread, to distinguish recurrent tumor from posttreatment changes, and to monitor the result and response of treatment. Cross-sectional imaging supplements and compliments the physical examination by delineating the complex anatomic and pathological changes of the neck. CT and MRI complement each other; certain conditions are better studied with one than the other. Various strengths and weaknesses of each modality should be carefully considered when selecting them for tumor assessment and follow up. Certain newer techniques, such as CT and MR perfusion, MR spectroscopy, facilitate the evaluation of functional parameters in oncologic patients, such as tissue perfusion, which can integrate the morphologic and metabolic information derived from conventional techniques and have the potential to identify the characteristics that could indicate malignant progression. Recently, functional imaging with 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) was introduced and found to be superior to conventional imaging work-ups in the evaluation of patients with head and neck malignancies. It improves the detection of occult cervical lymphatic disease, distant metastasis, and assists in localization of unknown primary carcinoma of head and neck region or a synchronous second tumor. Combined G.V. Shah (*) Radiology Department, University of Michigan, B2A209, UM11S, 1500 E. Med Ctr Dr., Ann Arbor, MI 48109, USA e-mail: [email protected]

PET/CT scanners have improved anatomic localization of HNC, incorporating the anatomic accuracy of CT with functional data of 18FDG-PET. From methodological development, these morphologic investigations are making the critical transition to preclinical and clinical validating methods and eventually to widespread clinical tools. Keywords  Head and neck cancer • Staging • CT perfusion • MR perfusion • MR spectroscopy • PET/CT • Squamous cell cancer • Synchronous second tumor • Unknown primary

A head and neck cancer (HNC) accounts for 3–5% of all the malignant conditions worldwide and is the fifth most common cancer condition [1]. HNC encompasses a variety of cancers, 90% of which are head and neck squamous cell cancers (HNSCCs), arising from a variety of sites. The primary risk factors for HNSCC in American men and women are tobacco and alcohol use. In 2009, approximately 83,730 new diagnoses and 18,860 deaths were expected in United States due to HNC [2]. Patients with HNSCC require a careful evaluation and a multidisciplinary team approach to determine optimal management. Treatment planning depends to a large extent on TNM staging, which is evaluated with physical examination, endoscopies, and cross-sectional imaging [3]. Radiologic imaging with CT and MR imaging is extensively utilized to evaluate soft tissue masses of head and neck. These masses are diagnosed and staged primarily on the basis of physical examination and CT and MRI findings [4–6]. Imaging has become a vital and integral tool in characterizing and staging of malignant tumors involving the head and neck. CT and MRI provide essential information about the deep extension of clinically detected masses and also delineate additional clinically unsuspected masses [7, 8]. Accurate staging at the time of diagnosis is critical for the selection of appropriate treatment strategy. Precise prediction of the extent of primary tumors, cervical lymph node status, and distant metastatic spread is important for treatment planning and prognosis. The goal of imaging in patients with HNC is to establish tumor extent and size, to assess nodal disease, for possible perineural tumor spread, and to distinguish recurrent

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tumor from posttreatment changes [9]. Imaging is also essential to follow up the patients after various therapeutic options available for the treatment are exercised, including surgery with or without radical dissection, lymph node dissections of various severities, radiotherapy, chemotherapy, and various combinations of all these [10]. Accurate evaluation of all these factors prior to treatment helps guide surgical extent or radiation porta, minimizing locoregional treatment failure. CT and MRI are the most commonly utilized imaging modalities for the assessment of primary malignant tumor, local extension, and lymph nodal involvement. They are also the first imaging modalities for monitoring the result and response of surgical intervention, radiation or chemotherapy, or combinations thereof. In this goal, cross-sectional imaging supplements and compliments the physical examination by delineating the anatomy and pathological changes of the neck. Complex anatomic structures and regions, such as the orbit, skull base, paranasal sinuses, deep spaces of the suprahyoid and infrahyoid neck, larynx, and lymph nodes, require that the radiologist be familiar with the imaging modalities available and their appropriate applications. CT and MRI complement each other; certain conditions are better studied with one than the other. Various strengths and weaknesses of each modality should be carefully considered when selecting them for tumor assessment and follow up [11]. The interpretation of CT and MRI should be based on the patient’s history, physical findings, comorbidities, and previous procedures that may influence the structures visualized. Comparison with previous imaging is also essential to reliably understand the present condition.

Standard CT Computerized tomography (CT) was introduced in the 1970s and revolutionized body imaging. It is inexpensive, fast, and ubiquitous in most medical centers. CT is quite good at delin-

Fig. 13.1  Axial postcontrast CT scan showing T3 stage right aryepiglottic fold carcinoma (a) with transglottic extension (arrow) and metastatic right level 2 lymphadenopathy (b) consistent with N1 disease (arrowhead)

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eating tumor extent and nodal disease. In head and neck tumors including squamous cell carcinoma, CT helped in tumor staging, which dictated patient management and related to prognosis [8]. Helical multidetector computerized tomography (MDCT) with 16 and now 64 detector rings has rapidly now become the new industry standard in CT imaging. This along with dynamic acquisition typically has resulted in reduced scan time, thinner sections, increased anatomic coverage and better resolution of reformatted images, and threedimensional reconstruction. Section thickness as low as 05 mm can be achieved along with acquisition of up to eight images per second [12]. This has greatly enhanced the sensitivity and specificity of CT scan in HNC for primary staging as well as posttherapeutic follow-up (Fig. 13.1). The anatomic coverage of a neck CT should include the base of the skull and should extend up to the medial end of the clavicles with 4-mm thick slices. Additionally, 2  mm slices and higher zoom factor may be employed at the region of interest using reconstructed spiral data. In patients with significant dental hardware, additional angulated images may also be obtained for better anatomic coverage avoiding streak artifacts. CT has proved to be a modality of choice for initial workup of a patient suspected of HNC and proved excellent for initial locoregional and lymph nodal staging and for posttherapeutic follow-up.

CT Perfusion CT perfusion (CTP) facilitates the evaluation of functional parameters in oncologic patients, such as tissue perfusion, which can integrate the morphologic information derived from conventional CT techniques. It is a dynamic contrastenhanced technique which is used for quantitative assessment of tissue microcirculation [13], and it has recently been rediscovered as a promising noninvasive tool for the evaluation

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of the microcirculatory changes associated with several neoplasm, including cancers of the head and neck [14–17]. CTP technique is based on the central volume principle, which relates blood flow, blood volume, and MTT as: blood flow (BF) = blood volume (BV)/MTT. Faggioni et  al. have shown that BV, BF, and permeability-surface area product are significantly higher, whereas MTT is significantly reduced in head and neck tumor (both primary neoplasm and lymph node metastases, whenever present) compared with normal tissue and with muscle taken as a reference (p < 0.01); moreover, the alteration of CTP parameters correlates with histopathologic diagnosis of adenocarcinoma in all cases [14]. Ash et al. have shown that CTP parameters of the neck (BF and BV) correlate positively with microvessel density (MVD) of endoscopic biopsy specimens obtained from primary tumor sites of HNSCC [18]. Although, it seems unlikely that CT perfusion will replace biopsy for pretreatment assessment of MVD, CTP has the potential to monitor treatment response by enabling noninvasive assessment of alterations in MVD and acting as a surrogate marker for tumor oxygenation.

Standard Magnetic Resonance Imaging Magnetic resonance imaging (MRI) introduced in the 1980s was a quantum jump in diagnostic imaging of the head and neck pathologies. Some of the earliest investigations in head and neck imaging with MRI highlighted the ability of MRI to differentiate neoplastic from inflammatory lesions. MRI provides essential information about the deep extension of clinically detected masses and also delineates additional clinically unsuspected lesions [7]. It has added value for the detection of soft tissue extent, marrow involvement, and perineural spread [19]. The excellent tissue characterization and noninvasive multiplanar imaging capability of MR imaging result in more accurate diagnosis of neoplastic and benign tumors of the head and neck [20–24]. MRI is reported to be superior to CT in detecting tumor extensions, in separation of edema from the tumor, and in the evaluation of possible bone marrow invasion. Dynamic MRI is also utilized to plan and evaluate radiotherapy of HNC [25]. MRI of the neck should be tailored for the anatomic region and process under evaluation. A standard head coil usually suffices for relatively localized examinations of the suprahyoid region and base of the skull, whereas, the infrahyoid neck requires a neck coil. Axial, coronal, and sagittal sequences are essential. Unenhanced axial T1-weighted images display anatomic relationships and can detect lesions (e.g., lymph node lesions) embedded within fat. T1-weighted coronal images can define the false vocal cords, true vocal cords, laryngeal ventricle, and the floor of mouth [26, 27].

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T1-weighted sagittal images provide helpful information about the preepiglottic space and nasopharynx. T2-weighted transaxial images characterize tissue, detect tumor within muscle, demonstrate cysts, and assist differentiation of posttherapy fibrosis from recurrent tumor [28]. Gradient moment nulling, flow compensation, cardiac gating, and presaturation pulses are some techniques used to minimize motion artifacts [26]. Gadolinium (Gd)-enhanced images improve delineation of margins in many lesions. Fatsuppression techniques, such as short tau inversion recovery (STIR) and frequency-selected fat suppression, may improve the conspicuity of soft-tissue lesions embedded in fatty tissue by selectively diminishing the hyperintensity of fat on T1-weighted images [29] (Fig.  13.2). Postcontrast T1-weighted images usually best delineate the tumor margins [30], and this may be further improved with fat saturation (fat-sat), which, however, frequently results in artifacts and image degradation [31]. However, the normal enhancement of the aerodigestive mucosa may conceal small mucosal tumors. Early investigators credited MR imaging with greater precision in head and neck imaging than was warranted [32]. Conventional MR imaging did not have the last word in histological specificity, early detection of primary malignancy, and differentiating neoplastic from inflammatory lymph nodes. In spite of early enthusiasm, MR imaging did not eliminate the need for biopsies or aspirations of lesions. Spin echo imaging is still the mainstay of MR imaging, but now various new techniques hold promise for the future of head and neck imaging [33].

MR Diffusion Although diffusion-weighted MRI (DWI) has been used for long for the evaluation of brain pathology, its potential utility for evaluating extracranial neoplastic disease has only recently been recognized. Hypercellular tissue within malignant tumors will show low ADC values [34, 35], while tissue changes such as edema, inflammation, fibrosis, and necrosis show low cellularity and hence higher ADC values [33] (Fig. 13.3). Diffusion weighted imaging of oropharynx can easily be performed at the time of MR conventional imaging and adds approximately only 1–2 min of additional time to the examination. Localization and extent of primary squamous cell cancer, one of the commonest malignant neoplasms of head and neck, is usually well defined by CT or conventional MRI. High sensitivities and specificities, better than CT or conventional MRI are also reported in staging of neck lymph nodes in squamous cell carcinoma [36, 37]. Whole body DWI at high b-values with ADC mapping is technically feasible and improves assessment of metastatic spread in routine

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Fig. 13.2  CT-PET (a and b) images showing FDG avid nasopharyngeal mass (arrow head). Axial T2W (c) and pre- and postcontrast fat ­suppressed T1W images (d and e) showing enhancing mass within the

left posterior nasopharynx crossing to the right side. Fluid in the right mastoid air cells (arrow) secondary to Eustachian tube dysfunction

MR examinations. The characterization of neck lymph nodes remains a difficult issue with anatomy-based imaging methods, and DWI may be useful in this regard [38, 39]. DW imaging performed with ADC(b0 − 1,000) values had higher accuracy than turbo spin-echo MR imaging in nodal staging, providing added value in the detection of subcentimeter nodal metastases [39].

or volume of the head and neck lesions depending on underlying pathologic processes [33]. This technique has been previously studied in characterizing brain ischemia, particularly in identifying infarcted tissue versus tissue at risk [40]. Changes in perfusion characteristics are also demonstrated in neoplastic tissue (Fig. 13.4). Generally, these findings may not add substantial additional information regarding tumor extent at the diagnosis. However, such imaging may be of benefit in qualitative analysis of tumor tissue. Specifically, additional recent studies have demonstrated that squamous cell carcinomas of the upper aerodigestive tract with increased blood volume/flow are more chemosensitive than other lesions with relative decreased perfusion parameters. This is likely due to relative increased oxygenation and metabolism of such lesions [16]. Such perfusion techniques could be particularly useful in determining which patients would benefit from such medical treatment, as opposed to surgical therapies which may not always preserve organ function.

MR Perfusion Perfusion imaging evaluates dynamic microscopic blood flow changes through a region of interest. Changes in tissue signal intensity on MRI are measured during a dynamic contrast infusion. Blood flow, blood volume, and transit time parameters of tissue regions can be then generated. Perfusion characteristics of tissue demonstrate changes in blood flow

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Fig. 13.3  Axial DWI (a) showing restricted diffusion in a left masticator space adenoid cystic cancer (arrows) with low ADC values (b) as seen on corresponding ADC maps (arrows)

An additional area of interest is in regard to tumor recurrence or regression. Conventional MRI or CT may simply demonstrate increased contrast enhancement within the treated neck. However, morphologic changes in tissue appearance (such as increase in size or nodularity) may not be well demonstrated on early posttreatment conventional imaging. Recent studies have concluded that for recurrent oral cavity and oropharyngeal carcinomas, perfusion parameters are altered. Specifically, BV and BF within recurrent tumor tissue are elevated in comparison to therapy-altered tissue, with corresponding decreases in transit time [41]. Perfusion imaging, such as diffusion imaging, adds little time to either conventional MRI or CT examinations and can also be obtained noninvasively [42].

MR Magnetization Transfer Magnetization transfer (MT) technique may be useful for differentiating enhancing lesions from background tissue and defining poorly enhancing lesions. This technique is based on the principle that the selective magnetization of protons associated with macromolecules may be transferred to the water protons that constitutes the MT image. A strong MT effect is observed where an efficient transfer mechanism exists between the two proton populations. This is exploited to improve contrast between mass lesions that demonstrate an MT effect and background tissue like fat that does not [43]. Use of MT can improve contrast between head and neck lesions and background tissues. MT is shown to improve

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Fig.  13.4  (a, b, c) Large posterior oropharyngeal wall squamous cell carcinoma demonstrates increased DWI (a) and decreased ADC (b) signal intensity at presentation. Posttherapy, the lesion has decreased greatly in size (c). (d) Blood volume map of the same

patient as in images a–c demonstrates increased perfusion values of the lesion (circled) in comparison to the adjacent tissues at presentation (from [33], reprinted with permission from John Wiley and Sons)

depiction of enhancing lesions adjacent to tissues with a strong MT effect [44]. MT can also aid unenhanced MR imaging in the delineation of tumors or lymph nodes in the parotid gland. MT is not indicated for cystic lesions, because they are generally well shown on a T2-weighted image or for cervical lymphadenopathy within lipoid tissue, because that has natural tissue contrast on conventional MRI [44]. However, MT has not enjoyed widespread application in head and neck imaging, partly because conventional imaging usually provides sufficient delineation of most primary lesions and lymphadenopathy.

of the anatomic information provided by MRI [45]. 1H-MR spectroscopy has the potential to assess biochemical composition and hence identify characteristics that could indicate malignant progression. It has the unique ability to analyze the tissue at the molecular level by evaluating the presence of specific metabolites. This is especially helpful to characterize lesions that have equivocal features on standard anatomic imaging. Early metastatic infiltration of nonenlarged lymph nodes or residual malignant disease in patients undergoing treatment for malignant process may also have normal or ambiguous appearance on routine anatomic CT or MR imaging [46]. In the case of HNSCC, it has been shown that 1H-MR spectroscopy has the potential to differentiate between normal and malignant tissue with a high degree of sensitivity and specificity [45, 47–50] (Fig.  13.5). MR spectroscopy of HNC and lymph nodes helps to differentiate nonmalignant from malignant tumors and lymph nodes and also helps to differentiate between residual malignancies from postradiation changes. Elevation of the Cho/Cr ratio appears to be a consistent finding

MR Spectroscopy It is now widely accepted that cancer progression is accompanied by intracellular biochemical changes. Magnetic resonance spectroscopy (MRS) provides a noninvasive method for evaluation of various diseases of head and neck independent

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Fig. 13.5  Patient with throat pain and dry cough exhibits a nasopharyngeal mass on MR imaging. (a) T1 axial images show a large nasopharyngeal midline soft tissue mass with nonspecific features and without frank aggression. (b) 1H-MRS reveals attenuation of N-acetyl aspartate peak, elevation of choline peak, and increased choline to creatine ratio compatible with malignant mass. This lesion was proved on biopsy to be a squamous cell carcinoma (from [45], reprinted with permission from Elsevier)

for HNSCCA and has also been identified in analysis of various SCCA cell cultures and SCCA containing cervical metastatic lymph nodes [47]. Higher levels of choline metabolites in tumors are believed to be due to increased cell proliferation

and biosynthesis, while reduced creatinine resonance likely reflect increased energy metabolism within tumors [51]. For prognostication, MR spectroscopy has the potential to contribute to an accurate and early prediction of tumor behavior

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and response to treatment in squamous cell carcinoma of the head and neck region. Using the choline-to-creatine (3.2/3.0  ppm) and the 1.3/0.9  ppm spectral intensity ratios (signal due to lipid or lactic acid), a sensitivity of 83% and a specificity of 82% were obtained in predicting which HNC patients would fail treatment [52]. Tumor hypoxia is a common phenomenon in solid tumors and has been shown to adversely affect the treatment outcomes in patients with head and neck (HN) squamous cell carcinoma treated with conventional therapy [53–55]. Resonance from lactate (Lac, 1.3 ppm) may be a marker for tumor oxygenation and may help staging and was thought to have potential for staging and monitoring the treatment [56]. However, in a recent work the lactate SI did not correlate with tumor pO2, treatment response, or locoregional control in a series of 62 patients with resectable stage IV HN squamous cell carcinoma undergoing induction chemotherapy [57]. Additional research is needed to refine this technique.

PET/CT Conventional morphological imaging methods such as CT and MRI have been the mainstay of diagnostic work-up for diagnosis, staging and posttherapeutic follow-up in patients with HNC. Recently, functional imaging with 18 F-fluorodeoxyglucose (FDG) positron emission tomography (PET) was introduced and found to be superior to conventional imaging work-ups in the evaluation of patients with head and neck malignancies [58–60]. 18FDG-PET has higher sensitivity and specificity for detecting lymph node

Fig. 13.6  Mantle cell lymphoma showing FDG avidity (a) in a nonenlarged left level 1 lymph node (arrows) in the neck (b)

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metastases than CT or MRI. It improves detection of occult cervical lymphatic disease, distant metastasis, and assists in localization of unknown primary carcinoma of head and neck region [61–65]. 18FDG-PET is considered superior to CT and MRI for local staging and detection of malignant characteristics in cervical lymph nodal enlargements [58, 59, 66–69]. It has a high negative predictive value (NPV) of approximately 90%, which is more than any other imaging modality. There is growing evidence that 18FDG-PET imaging is increasingly accepted as a valuable imaging tool in the evaluation of patients with head and neck carcinomas [61–64, 70–73]. The potential clinical applications include pretreatment staging, treatment monitoring, and evaluation of the previously treated patients [74] (Fig. 13.6). Poor quality of anatomical localization of the primary tumor and metastases on 18FDG-PET can have negative impact on staging and management [75]. The poor spatial resolution of 18FDG-PET is a limiting factor, especially within the intricate anatomy of head and neck [68]. Combined PET/CT scanners overcome these limitations by fusing the anatomic data of CT with functional data of 18FDG-PET [76–78]. In PET/CT, the most relevant additional effect is that the CT data adds specificity to 18FDG-PET data [79, 80]. The utility of PET/CT has been evaluated extensively in head and neck neoplasm. Several of these studies showed that the integrated combination of CT and 18FDG-PET is more accurate than either of the modalities alone for detection and anatomic localization of HNC, thus enhancing the patient care [81–86]. The accuracy of integrated PET/CT is also more than 18FDG-PET and CT images viewed side by side [82, 87–90]. In one study, CT data improved the specificity of the images in approximately two-third of patients with lesions

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seen on 18FDG-PET images [91]. In some situations, such as very small disseminated pulmonary metastases, addition of CT is able to increase the specificity and also the sensitivity of PET/CT examination [80]. PET/CT can detect unknown primary tumors of the upper aerodigestive tract [92, 93]. PET/CT can detect primary squamous cell carcinoma in 30–50% of patients presenting with an unknown primary tumor. PET/CT is generally performed after confirming the presence of metastatic squamous cell carcinoma. It is usually performed before endoscopic biopsies to improve the tissue yield. This diagnostic yield can increase with PET/CT because as it improves the anatomic localization of areas of abnormal FDG uptake [94, 95]. PET/CT is also utilized for determining response to chemotherapy and/or radiation. Comparison of pretreatment standard uptake values (SUVs) to SUVs 2 weeks into treatment can allow measurement of the speed of response and also the sensitivity of the tumor to the treatment technique [96]. Poorly responsive tumors can then be treated to higher effective tumor doses of radiation, or surgery can be performed. Initial results suggest that PET/CT can be used to assist in defining primary site and nodal tumor targets for radiation therapy approaches. PET/CT is useful adjuvant to clinical staging of squamous cell carcinoma and its utilization will increase with advancement of technology.

Local Tumor Detection and Staging The most important information required before surgery for proper therapeutic planning is the accurate knowledge of location, size, extent, the depth of invasion of the primary tumor, and its relation to the surrounding structures [68, 97]. Large primary tumors of the oral cavity or the oropharynx can be detected easily by clinical examination. The sensitivity of FDG-PET was considered even higher than CT or MRI for detection of primary tumors [98]. The sensitivity of FDGPET for detection of primary carcinoma ranged from 88 to 100% [60, 62, 99, 100]. Both MRI and CT can provide additional information about tumor extension into the deep spaces, the relationship to adjacent structures, and bone infiltration needed for treatment planning. Sensitivity of MRI earlier was thought to be less than that of CT [61, 99]. However, with increased technical improvements, it is thought to be comparable to CT [3]. Scattering of focal uptake in primary oropharyngeal tumors can lead to overestimation of the extent of primary disease and physiologic uptake in oropharynx may obscure small primary tumors in oropharynx [101]. Thus FDG-PET alone cannot provide the detailed information needed for planning of tumor resection, but fusion of FDG-PET data with CT data in PET/CT can overcome this limitation.

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Sensitivity of CT, especially in oropharynx can be compromised by streak artifacts from dental hardware, especially if the size of the tumor is small [3]. However, high metabolism on FDG-PET would indicate the possibility of an underlying mass (Fig.  13.7). Earlier, the sensitivity of MRI was thought to be less than that of CT [61, 99], but with increased technical improvements, it is thought to be comparable to CT [3]. Some of the earlier reports showed that FDG-PET was more accurate than CT or MRI for local detection of smaller tumors [61, 99, 100]. But some more recent studies have shown that CT and FDG-PET are equivalent in local staging [60, 102]. CT detects lytic foci of cortical mandibular invasion, which are best accomplished with a dedicated dental protocol. The reported sensitivity and specificity for standard neck CT in the detection of mandibular involvement are 96 and 87%, respectively (Fig. 13.8) [103]. However, a later study demonstrated a 93% accuracy of MRI in detecting mandibular involvement in patients with oral and oropharyngeal cancer [104], indicating that CT may not be necessary to evaluate for cortical invasion. MRI with contrast-enhanced T1-weighted fat-sat images, provides satisfactory accuracy of tumor thickness. Presence of malignant neoplasm adjacent to the neurovascular bundle is highly concerning for invasion. Tumors larger than 2 cm with aggressive margins and deep sublingual extension probably involve the neurovascular bundle [30]. Oral malignancies, especially of buccal spaces and retromolar trigone are better visualized using the “puffed cheek” CT technique, in which the patients perform a modified Valsalva maneuver during the scan distending the oral cavity by air [105]. Deep extension of nasopharyngeal cancer including the presence of skull base invasion and intracranial spread is better evaluated with MRI than CT [106, 107]. Skull base invasion may occur through the neural foramina by perineural tumor spread, which primarily occurs after invasion of the pterygopalatine fossa, foramen ovale, and hypoglossal canal [108] (Fig.  13.9). Nonenhanced T1-weighted images are very well suited to evaluate perineural extension, revealing homogeneous gray mass of tumor that against natural tissue contrast of T1 bright fat planes and bone marrow. Pre- and postcontrast T1-weighted MRI is very accurate in the detection of subtle perineural tumor extension. Evaluation of possible perineural spread should be performed in all patients with facial paralysis and facial pain or numbness, because these symptoms may be the initial presentation of a head and neck malignancy [109, 110] (Fig.  13.10). Complementary direct coronal CT images with bone algorithm are recommended to evaluate subtle bone erosion which may escape detection by MRI. Cartilage invasion by laryngeal and hypopharyngeal tumors is an important imaging finding because it automatically leads to a T4 stage [9]. The overall sensitivity is 82%,

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overall specificity is 79%, and overall negative predictive value of cartilage erosion on CT overall is 91% [111]. Cartilage invasion on MRI shows high T2 signal intensity, a low to intermediate T1 signal, and postcontrast enhancement. However, due to frequent reactive inflammation, edema, and fibrosis, the MRI findings of cartilage invasion may frequently be false-positive, resulting in a positive predictive value of only 68–71% [112]. However, the advantages of MRI over CT for soft tissue differentiation may be outweighed by motion artifacts. CT remains a valuable and

Fig. 13.7  (A) a, b: Axial postcontrast CT scan (a) showing dense streak artifacts from unmovable dental hardware obscuring FDG-avid squamous cell cancer in the oral tongue (arrow) with metastatic left level 2 lymph node (arrow head) as seen on PET scan (b). (B) a, b: Axial postcontrast CT scan (a) showing large necrotic left level 2 lymph node (large arrow) and necrotic left level 5 lymph node (small arrow), with FDG avidity on the corresponding PET scan (b). (C) a, b, c: CT thorax in mediastinal windows (a), and lung windows (b) showing a metachronous lung cancer (arrow) with increased FDG uptake on PET scan (c)

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frequently used screening modality for the larynx as it is fast and readily available. Imaging studies cannot reliably distinguish benign from malignant salivary gland masses. MRI is the modality of choice for evaluation of parotid masses [20]. The real advantage of cross-sectional imaging is the ability to accurately reveal the location and extension of a tumor and to assess for perineural tumor spread. Magnetization transfer, dynamic imaging, and especially, diffusion imaging have shown promising results in the detection of parotid malignancies [113].

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Lymph Node Staging

Fig. 13.7  (continued)

The relationship of a tumor to the facial nerve is difficult to determine on MRI. However, the lateral margin of the retromandibular vein on cross-sectional imaging as a marker for the facial nerve has an accuracy of approximately 90% [114]. A careful search for perineural tumor spread along the facial, auriculotemporal, and mandibular (V3) nerves should be undertaken on MRI scans in all patients with parotid masses [115]. Multiple series have been reported evaluating FDG-PET or PET/CT for patient with newly diagnosed HNSCC in the preoperative setting [59, 62, 116]. Sensitivity of FDG-PET was reported to be 98% and of PET/CT 97% for the detection of primary tumors in patients with newly diagnosed HNSCC in a large series with 167 patients [68], higher than sensitivity of CT (86%) and MRI (88%) in the same patient set. Similar results were reported in numerous previous studies [58, 59, 62, 81, 84, 85, 116]. Even as sensitivity of PET/ CT is considered higher than any morphological imaging for primary detection of HNSCC, the detailed anatomic information such as depth of invasion and relationship of tumor to surrounding structures could not be provided only by the CT data of PET/CT. This may be due to inherent technical limitations of CT data set. With the availability of multislice and multidetector scanner capability in future with PET/CT, this situation may improve.

As most primary head and neck malignant neoplasm have a relatively high incidence of nodal metastasis, the staging of neck is most important before a therapeutic plan is evolved. Staging can be done by a combination of clinical palpation and anatomic imaging. Nearly 40% of all lymph nodes in the body are located above the clavicles. Lymph nodes are usually embedded within the fat planes that surround the vessels and separate major cervical muscles. Therefore, the fat of the neck provides an excellent natural contrast with the nodes on T1-weighted MR images [11]. Lymph nodes are divided into ten major groups [117] named for the structures in proximity to nodal location. Patients with limited nodal spread of HNC are often treated surgical with radical neck dissection; while more extensive disease may additionally require adjuvant radiation therapy. Complete removal of all metastases lymph nodes is essential for curative treatment. Lymph node metastases are common in patients with HNCs. In up to 20–30% of patients, lymph nodal spread of the disease is found, even though it may not be apparent on physical exam [118, 119]. The prognosis for these patients is strongly influenced by the presence of lymph node metastases [99]. Metastatic lymph node disease was found in approximately 50% of the patients at the time of diagnosis [3, 70]. The imaging recommendations are mixed regarding an appropriate modality for evaluating lymphadenopathy [5, 120, 121]. CT is preferred because of its availability, speed, and excellent spatial resolution. Lymph nodes are usually embedded within fat, and fat is well portrayed by CT (see Fig. 13.6). MRI has superior soft-tissue contrast and multiplanar capabilities. CT and MRI, have a high rate of falsenegative diagnoses, which can be explained by micrometastases within otherwise normal lymph nodes [31, 122]. The reported sensitivity for CT in the detection of metastatic lymph nodes is from 67 to 90% [31, 70, 122–124] and for MRI is from 71 to 91% [3, 31, 64, 70, 99, 122]. The reported sensitivities of PET for nodal disease range from 67 to 91% [3, 61, 64, 70, 71, 100, 102, 123, 124]. Both FDG-PET and PET/CT have technical resolution limitations of 4–5 mm and were unable to detect lymph metastases smaller than 4–5 mm, contributing to false-negative results [125–127]. The reported specificity of FDG-PET ranges from 88 to 100% [64, 70, 71, 83, 100, 123]. The specificity value for CT is 38–97% and for MRI is 48–94% [31, 70, 123, 128]. False-positive FDG-PET findings may be primarily due to its inability to discriminate between inflammatory process and tumor infiltration [98]. This is because FDG is not tumor specific tracer but a metabolic marker and hence various inflammatory processes can lead to increased FDG uptake, potentially returning false-positive results [129]. However, a practical benefit of employing PET/CT in presurgical evaluation for

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Fig. 13.8  Axial postcontrast CT scan (a) showing stage T4 left retromolar trigone cancer (arrow) with the destruction of left mandibular ramus (arrow head) on bone windows (b). Perineural spread along left inferior alveolar nerve with loss of normal fat in the alveolar foramen on the left (d), compare with normal right side (c) (arrowhead)

lymph node staging in patient with HNSCC is improved imaging staging for the expert and also a nonexpert interpreter [83]. PET/CT imaging is also reported to reduce equivocal head and neck image interpretations and increase evaluator confidence [130]. Combining structural information with morphological imaging such as CT and metabolic information with functional imaging such as FDG-PET with coregistered PET/CT is a method of choice for lymph node imaging in the future.

Distant Metastases Distant metastasis to other organs and distant lymph nodes from HNSCC is generally a late event and usually represents an incurable disease [131]. Lung is the most common site of distant spread; however, distant bone metastasis can also occur in case of other widespread metastatic disease [132, 133]

and can cause severe local morbidity at the metastatic site [134]. The reported incidence for distant bone metastases in HNSCC ranges from 17 to 31% [135–137]. Apart from lungs, screening for distant metastases is routinely not performed in initial staging of patients with HNSCC [132, 138]. However, some studies have shown FDG-PET to be valuable in detecting distant metastasis in advanced HNSCC, suggesting a role for whole body FDG-PET scanning, including lungs and bones for initial staging [139–141]. PET-CT may be performed in squamous cell carcinoma to evaluate for possible occult distant metastases to the lungs or bones [123] (see Fig. 13.7). The presence of pulmonary metastases upstages a patient from M0 to M1 and alters treatment regimen. Routine imaging work-up for patient with squamous cell carcinoma pulmonary includes conventional radiography of the chest at most institutions. Chest CT is performed in patients with advanced stage disease. A solitary nodule on CT scan may represent a metastasis or

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Fig. 13.9  Coronal fat suppressed postcontrast T1-W images showing large infiltrating soft tissue attenuation mass in the left masticator space (bold arrow) extending into the pterygopalatine fossa (a). There is associated abnormal enhancement along the second and third divisions of the left trigeminal nerves and left vidian canal (small arrows) (a, b, d). There is infiltration of the left orbital floor with enhancing soft tissue and thickening of the left inferior rectus muscle (small arrow) (c)

a granuloma. PET would be helpful in this evaluation as a FDG-positive nodule would likely be metastatic and may require biopsy. An FDG-negative nodule may likely indicate a granuloma.

Unknown Primary Tumor The incidence of unknown primary tumors in the head and neck region ranges overall from 3 to 7% of all HNC including HNSCC [63, 66, 128, 142–148]. Apart from the routine physical examination, the evaluation includes fiberoptic laryngoscopy/nasopharyngoscopy, panendoscopy, and morphological imaging, including CT and MRI and directed biopsy [142, 146, 147]. The areas most likely to harbor an occult primary, such as tonsil, tongue, base, piriform fossa, and

postnasal space should be thoroughly evaluated with physical examination and office-based endoscopies [147]. Focused morphological imaging with CT and MRI looking for evidence of primary as well as additional areas of lymphadenopathy are also performed. Further management is often a combination of surgery and radiotherapy; however, this depends on the primary site of the disease as well as the treating center [149, 150]. In spite of thorough clinical, endoscopic and morphological imaging, 1–2% of HNC patients will not have a primary site detected [151, 152]. An important application of PET imaging may be in patients with nodal disease and unknown primary tumor – the primary site has been found in 10–60% of cases when conventional imaging and clinical investigations have failed [9]. FDG-PET is generally more sensitive than morphologic imaging in patients with unknown sites of the primary carcinoma [153, 154]. However, it is also associated with

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Fig. 13.10  Postradiotherapy “Facial Neuritis.” Axial (3-mm section) postcontrast, fat suppressed T1W image showing increased enhancement of the tympanic segment of the left facial nerve (arrow), compared to normal right-sided facial nerve (arrow head)

false-positive findings in up to 11% of these cases [153, 155]. Tumors of oral cavity account for a majority of cases with unknown primary and can generally be detected by clinical examination. However, in head and neck regions with lower sensitivity for clinical examinations and morphological imaging, the role of FDG-PET and PET/CT becomes more evident [3]. Tumor detection rate of about 31% of primary tumors is reported in patients presenting with unknown primary [148]. A few retrospective studies suggest FDG-PET detection rates of 24–27% for an occult head and neck primary carcinoma [63, 156]. Another study reported a low rate of true positive scan (33%) but a high rate of true negative scans (88%) [157], suggesting that negative FDG-PET or PET/CT helps to rule out a primary site (Fig. 13.11). This is complicated by the fact that false-positive reports are reported in large lymph nodes up to 20 mm in size [122, 123] or in necrotic lymph nodes. PET/CT serves as a valuable clinical tool for occult metastatic disease of head and neck, most commonly HNSCC and synchronous primary tumors.

Fig. 13.11  Patient presented with bilateral lymph nodal neck masses. PET/CT reveals unknown primary neoplasm of nasopharyngeal squamous cell carcinoma. (a) MIP PET image demonstrates bilateral increased abnormal FDG uptake in the neck (black arrows). (b) Axial PET in the region of nasopharynx shows focal abnormal FDG uptake in the region of the right torus tubarius (black arrow). (c) Axial CT of nasopharynx shows mild soft tissue fullness in the same region (white arrow). (d) Axial PET/CT demonstrates increased abnormal FDG uptake in the region of mild soft tissue fullness representing primary

unsuspected squamous cell carcinoma of nasopharynx. (e) Axial PET of neck at the level of mandibular angle demonstrates FDG uptake in bilateral level II cervical lymph nodes (black arrows). (f) Axial CT shows enlarged bilateral level II lymph nodes (white arrows). (g) Axial PET/CT demonstrates fusion imaging signifying malignant nature of enlarged lymph nodes (from Shah GV, Wong KK, Gandhi D, Parmar H, Mukherji SK. Squamous cell carcinoma: initial diagnosis and staging with PET/CT. PET Clin. 2007;2(4):469–80, reprinted with permission from Elsevier)

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Synchronous Second Tumor Patients with head and neck tumors also have a high incidence of secondary tumors of the aerodigestive tract (estimated at approximately 8%), and PET identifies synchronous primary neoplasms that are missed on conventional imaging. The incidence for metastatic spread to lungs in patients with HNSCC is low but there is also a high incidence of second primary tumor in a patients with HNC, with detectable lung lesion [158]. A few previous studies have shown a high sensitivity of 100% and positive predictive value of 85% for FDG-PET to differentiate a malignant from a benign pulmonary lesion [139, 159]. Due to its ability to conduct whole body imaging, PET/CT can be useful for the detection of distant metastases and second primary cancer (see Fig. 13.7) [160, 161]. PET/CT can serve as an excellent screening tool for distant metastatic disease or a synchronous primary tumor in the lungs [148]. In conclusion, morphologic imaging techniques are crucial for therapy planning in head and neck neoplasm. The highest sensitivity and optimal anatomic information of the local tumor site for local staging are provided by MRI. MRI, CT, and PET are similar for the detection of abnormal and pathologic lymph nodes. However, in case of equivocal findings by MRI or CT, PET provides relevant information for determining the extent of surgical neck dissection. FDG-PET and CT compliment each of the strengths, providing additional accuracy for staging HNC and make a notable impact on clinical decision making.

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13  Morphologic Investigations in Head and Neck Cancer primary head and neck carcinoma. Arch Otolaryngol Head Neck Surg. 2005;131(7):626–9. 149. Davidson BJ, Spiro RH, Patel S, Patel K, Shah JP. Cervical metastases of occult origin: the impact of combined modality therapy. Am J Surg. 1994;168(5):395–9. 150. Talmi YP, Wolf GT, Hazuka M, Krause CJ. Unknown primary of the head and neck. J Laryngol Otol. 1996;110(4):353–6. 151. Nieder C, Gregoire V, Ang KK. Cervical lymph node metastases from occult squamous cell carcinoma: cut down a tree to get an apple? Int J Radiat Oncol Biol Phys. 2001;50(3):727–33. 152. Grau C, Johansen LV, Jakobsen J, Geertsen P, Andersen E, Jensen BB. Cervical lymph node metastases from unknown primary tumours. Results from a national survey by the Danish Society for Head and Neck Oncology. Radiother Oncol. 2000;55(2):121–9. 153. AAssar OS, Fischbein NJ, Caputo GR, et al. Metastatic head and neck cancer: role and usefulness of FDG PET in locating occult primary tumors. Radiology. 1999;210(1):177–81. 154. Braams JW, Pruim J, Kole AC, et al. Detection of unknown primary head and neck tumors by positron emission tomography. Int J Oral Maxillofac Surg. 1997;26(2):112–5. 155. Bohuslavizki KH, Klutmann S, Kroger S, et al. FDG PET detection of unknown primary tumors. J Nucl Med. 2000;41(5):816–22.

219 156. Johansen J, Eigtved A, Buchwald C, Theilgaard SA, Hansen HS. Implication of 18F-fluoro-2-deoxy-D-glucose positron emission tomography on management of carcinoma of unknown primary in the head and neck: a Danish cohort study. Laryngoscope. 2002;112(11):2009–14. 157. Silva P, Hulse P, Sykes AJ, et al. Should FDG-PET scanning be routinely used for patients with an unknown head and neck squamous primary? J Laryngol Otol. 2007;121(2):149–53. 158. Tepperman BS, Fitzpatrick PJ. Second respiratory and upper digestive tract cancers after oral cancer. Lancet. 1981;2(8246): 547–9. 159. Wax MK, Myers LL, Gona JM, Husain SS, Nabi HA. The role of positron emission tomography in the evaluation of the N-positive neck. Otolaryngol Head Neck Surg. 2003;129(3):163–7. 160. Choi JY, Lee KS, Kwon OJ, et al. Improved detection of second primary cancer using integrated [18F] fluorodeoxyglucose positron emission tomography and computed tomography for initial tumor staging. J Clin Oncol. 2005;23(30):7654–9. 161. Kamel EM, Thumshirn M, Truninger K, et al. Significance of incidental 18F-FDG accumulations in the gastrointestinal tract in PET/CT: correlation with endoscopic and histopathologic results. J Nucl Med. 2004;45(11):1804–10.

Chapter 14

Ultrasound Investigations in Head and Neck Cancer Patients Yolanda Y.P. Lee, K.T. Wong, Ann D. King, and Anil T. Ahuja

Abstract  The wide availability, inexpensiveness, and its nonionizing nature make ultrasound an ideal initial imaging investigation in patients with head and neck cancer. Its high sensitivity and specificity (when combined with a guided FNAC) makes it a useful tool for cervical lymph node ­staging and investigating thyroid and salivary gland tumors. Gray scale ultrasound evaluates the internal architecture and local extent of superficially located head and neck cancers and color Doppler examines the tumor vascularity. Advances in elastography and contrast ultrasound further enhance the diagnostic capability of ultrasound. In addition, following treatment of head and neck cancer, ultrasound is a useful and safe tool for disease surveillance and assessment of treatment response. Keywords  Ultrasound • Head and neck cancer • Lymph node

Introduction Ultrasound (US) has a well-recognized role in imaging of patients with head and neck cancer. Its nonionizing nature, high sensitivity, and specificity (when combined with a guided FNAC) make it a useful tool for cervical lymph node staging and investigating thyroid and salivary gland tumors. In addition, US is superior to CT or MR in the resolution of superficial structures and provides detailed information of the internal architecture, vascular pattern, and local extent of superficially located tumors (thyroid, superficial salivary glands, and lymph nodes). Therefore, the major applications for ultrasound in the head and neck cancer include characterization of neck masses, guide FNAC/ biopsy, evaluate nodal status to accurately stage cancer, and

A.T. Ahuja (*) Department of Diagnostic Radiology and Organ Imaging, The Chinese University of Hong Kong, Hong Kong, China e-mail: [email protected]

follow-up patients postoperatively to exclude local or regional tumor recurrence [1]. Detailed sonographic appearance of all thyroid, salivary gland cancers, malignant lymph nodes, and their benign mimics in the head and neck is beyond the scope of this chapter. The following paragraphs discuss the principles and practical application of US (+FNAC) in evaluating these sites in the head and neck.

Role of Ultrasound in Thyroid Cancer Thyroid nodules pose a treatment dilemma as the prevalence of palpable nodules is 1–5% in iodine-sufficient parts of the world [2, 3]. The increasing use of high-resolution US in the head and neck detects thyroid nodules in 19–67% of randomly selected individuals [4]. The spectrum of these thyroid nodules ranges from the common multinodular change to malignant thyroid tumors that occur in 5–10% depending on age, gender, previous radiation history, and other factors [5, 6]. It is therefore necessary to identify the small group of patients with malignant thyroid disease so that prompt and appropriate treatment can be instituted; while avoiding unnecessary imaging and treatment in the vast majority with benign nodules. The management guidelines for patients with thyroid nodules and differentiated thyroid cancer is well established [7]. The mainstay of initial investigations include US (gray scale [GS], and power Doppler [PDS]), FNAC, and radionuclide thyroid scan. In patients with thyroid nodule >1–1.5 cm, an initial TSH level is obtained. If the TSH is subnormal a radionuclide scan is indicated to document whether the ­nodule is functioning. However, if the TSH is not suppressed a thyroid ultrasound is indicated. This is often combined with a FNAC and nodules FNAed based on their sonographic appearance rather than their size, as the US characteristics, such as echogenicity, microcalcifications, vascularity, are better than nodule size in predicting malignancy [8, 9].

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Sonographic Features that Help to Differentiate Malignant from Benign Tumors −− Echogenicity (Figs. 14.1–14.3): Hypoechoic thyroid nodules have an increased risk of malignancy. It represents microfollicular structure on histology, compared to macrofollicular lesions which tend to be iso/hyperechoic [10]. The risk of malignancy is 4% when the nodule is hyperechoic and this increases to 26% with hypoechoic nodules. However, ­echogenicity alone is a poor predicator of malignancy, ­specificity 49% and positive predictive value 40% [11]. −− Margins (Fig.  14.4): Malignant nodules are invasive by nature and tend to have irregular margins. −− Halo (Fig. 14.3): Benign hyperplastic nodules are slow growing, lack a true capsule, and displace adjacent vascularity.

Fig. 14.1  Transverse GS US (a) shows a solid, ill-defined, hypoechoic thyroid nodule (arrow). The corresponding PDS (b) shows marked intranodular vascularity (arrows). The overall appearances are suspicious of malignancy. Histology confirmed a follicular carcinoma. Open arrow: CCA

Fig. 14.2  Longitudinal GS US (a) shows a solid, fairly welldefined, hypoechoic, noncalcified thyroid nodule (arrows). Corres-ponding PDS (b) shows marked intratumoral vascularity (arrows). The combination of GS US and PDS suggests a malignant lesion which was confirmed at surgery

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Therefore, they demonstrate a “vascular halo” on color Doppler. Thyroid cancer may demonstrate an “avascular halo” on Doppler which represents the fibrous capsule around the tumor [12]. The absence of a halo has a specificity of 77% and sensitivity of 67% in predicting malignance [13]. −− Multinodularity: High-resolution US is far more sensitive than palpation in picking up small thyroid nodules. However, multinodularity does not bestow benignity on a thyroid nodule as patients with multiple thyroid nodules have the same risk of malignancy as those with solitary thyroid nodules [8, 14]. −− Cystic change (Figs. 14.5 and 14.6): True cysts of the thyroid gland are rare, and most “cystic” nodules seen on US are complex thyroid nodules with hemorrhage and necrosis. These complex nodules are predominantly cystic with

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Fig. 14.3  Transverse GS US shows a well-defined, partially haloed, solid, homogeneous, noncalcified thyroid nodule (arrows). Note its echogenicity is similar to the adjacent thyroid (asterisk). Hypoechoic solid nodules are suspicious for malignancy. The incidence of ­malignancy decreases as the echogenicity increases

Fig.  14.5  Transverse GS US shows a heterogenous cystic nodule (arrows) with intranodular septa (arrowheads), debris (asterisks), and comet tail artifact (open arrow) suggestive of colloid nodule. The debris is usually avascular on Doppler and is suggests of intranodular hemorrhage

Fig. 14.4  Longitudinal GS US shows a solid, ill-defined, hypoechoic thyroid nodule (arrows) with focal intranodular punctate calcification/microcalcification (arrowhead). Typical appearances of a papillary carcinoma

Fig. 14.6  Transverse GS US shows multiple, septated, heterogeneous nodules (arrows) with cystic change and comet tail artifacts (arrowheads) suggestive of colloid nodules in multinodular thyroid

i­nternal septa, and a “solid” component/debris which is often avascular and possibly represent blood clots. Pure cystic nodules have no risk of malignancy and complex, noncalcified nodules harbor a 3% risk of malignancy [15]. The

presence of a comet tail artifact is a good indicator of benignity and reflects condensed colloid within the nodule [16]. −− Calcification  (Figs. 14.4 and 14.7): Fine punctuate calcification (microcalcifications, <1  mm) which represent

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Fig. 14.7  Transverse GS US show a thyroid nodule (black arrow) with focal areas of dense calcifications (arrowheads) with posterior shadowing (white arrows) suggesting benignity. Curved arrow: CCA, open arrow: trachea

aggregates of psammoma bodies are seen in 25–40% of patients with papillary carcinoma [17]. As a sole predictor of malignancy it has an accuracy of 76%, specificity of 93%, positive predictive value of 70% [11], and also has good interobserver variability [18]. Coarse, dense shadowing calcifications are a reflection of fibrosis, necrosis, and tissue degeneration. Although often seen with benign nodules their presence with/without microcalcifications, in the center of a hypoechoic nodule are worrisome for malignancy [15, 19]. Curvilinear or “egg-shell” calcification was once ­considered as benign calcifications. However, interrupted rim calcification raises the possibility of malignancy [20]. −− Vascularity (Figs. 14.1b and 14.2b): Most benign nodules have absent intranodular vascularity and most malignancies have intranodular flow [8, 21]. However, as the ­negative predictive value is 88% a negative study does not eliminate the need for a biopsy [21, 22]. It has been reported that follicular nodules with no intranodular flow have a 3% probability of being malignant, compared to 15–20% likelihood in unselected follicular nodules. Vascular follicular nodules have a 50% probability of being malignant [23]. −− Shape: It has been reported anterior–posterior to transverse diameter (A/T ratio) ³ 1 (taller than wide nodule) has a sensitivity of 84% and specificity of 82% in the detection of a malignant nodule. −− Elastography: Is a technique that is beginning to be ­routinely used in the evaluation of thyroid nodules. It estimates the tissue stiffness on application of external force. Malignant nodules tend to be stiffer than benign nodules

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with increased tissue stiffness seen in malignant nodules compared to benign nodules [24, 25]. Recent reports have indicated that high elasticity scores were highly predictive of malignancy with a sensitivity of 97%, specificity 100%, positive predictive value of 100%, and negative predictive value of 98% [25]. −− Associated lymphadenopathy: US examination for ­thyroid nodules must include a detailed examination of the neck for lymph nodes as they are frequently seen in thyroid cancers and may alter management. Although most patient with thyroid cancer present with a thyroid nodule, 15–30% present clinically with an enlarged palpable node [26]. Thirty to forty percent of patients with papillary ­carcinoma have nodal metastases at presentation [27–29]. Follicular carcinomas show a lower incidence of cervical nodal metastases in the range of 10–15% [30]. Patients with medullary carcinoma, anaplastic carcinoma, ­lymphoma, and thyroid metastases also have a high ­incidence of adjacent nodal involvement [17, 31]. Nodes from thyroid cancers commonly involve the pretracheal, paratracheal, nodes, and those along the internal jugular vein. Metastatic nodes from papillary carcinoma have characteristic US appearances [32]: hyperechoic to adjacent muscle (80%), intranodal cystic necrosis (25%), and 50% show punctuate calcification (reflecting psammoma bodies). The metastatic nodes often resemble the primary thyroid tumor. Metastatic nodes from medullary cancer may also show intranodal calcification, but the nodes are usually hypoechoic and the calcification dense shadowing in type (reflecting amyloid deposition). −− Extrathyroid invasion: Although US is able to evaluate extrathyroid invasion, CT and MR better evaluate the spread of thyroid cancer to the larynx, trachea, and involvement of adjacent vessels [33]. Shadowing from the trachea makes US suboptimal in the evaluation of pretracheal, paratracheal, laryngeal, and tracheal involvement. The above limitation also applies to sonographic evaluation of malignant nodes at these sites. In evaluating the above sonographic features of thyroid nodules one must note that none of them alone are accurate in predicting malignancy. It is well accepted that US is a ­reliable predictor when multiple signs are present in the same nodule [13]. However, as the predictive value increases its sensitivity decreases [13]. The useful combinations to ­predict malignancy include: −− Microcalcifications and solid nature of the nodule [11] showed the highest accuracy (77%), specificity (96%), positive predictive value (75%) but a low specificity (30%). −− Absent halo combined with microcalcifications had a specificity of 93% but a sensitivity of 27% [13]. −− A combination of absent halo, intranodular flow, and microcalcifications had a specificity of 97% and a sensitivity of 16% [13].

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Role of Ultrasound in Recurrent Thyroid Disease The evaluation of a patient for recurrent tumor includes clinical examination, biochemical parameters, and imaging findings. The imaging modalities include US, CT, MR, and PET/CT. The postoperative distortion of anatomy makes US difficult but the superficial location of the recurrent tumors makes US (+FNAC) a useful examination as it clearly evaluates the thyroid bed and the neck for lymphadenopathy. Postoperative suture granulomas must not be mistaken for recurrent tumors in the thyroid bed. The granulomas are usually solid, hypoechoic, avascular/hypovascular, and may show dense shadowing foci within (sutures). A guided FNAC quickly establishes the nature of the lesion. CT and MR are easier to perform and have the added advantage that it is able to evaluate regional recurrence and any disease in the chest/mediastinum.

Role of Ultrasound in Salivary Gland Cancer Imaging plays an important role in the evaluation of these tumors and the various modalities have complementary roles. In many cases, US may suffice, in others it may be necessary to follow it with a CT/MR, and in some the role of US may be restricted to guiding a biopsy. Irrespective of the modality used imaging appearances are not a substitute for tissue diagnosis. The following limitations of US must be borne in mind when evaluating salivary gland cancers [34]:

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and establish the diagnosis by a guided FNAC (sensitivity 88–93%, specificity 75–99%) [35–37]. If the tissue diagnosis suggests a malignancy, an MR helps to evaluate deep ­extension of the tumor, perineural spread, bone infiltration, and the presence of deep seated nodes. MR is the initial investigation of choice for: −− Salivary gland mass with signs and symptoms suggestive of a malignant salivary gland tumor (short duration ­history, rapid enlargement of tumor, progressive facial paralysis, pain, trismus, or cranial nerve palsies associated with a salivary mass). −− Tumors arising from deep lobe of parotid gland, or large tumors bulging into the oral cavity. In this group of patients, the use of US is restricted to its assistance in image-guided biopsy. Compared to CT, MRI better delineates perineural spread, skull base involvement, parapharyngeal involvement, and minor salivary gland ­cancers [38]. However, in centers with no access to MR, CT may be used as it appears to have the same diagnostic value for salivary gland tumors [39].

Sonographic Features that Help to Differentiate Malignant from Benign Tumors −− Edge  (Figs.  14.8 and 14.9): Malignant tumors have illdefined edges, are irregular in outline compared to benign salivary gland tumors.

−− US does not adequately visualize the deep lobe of the parotid gland and the minor salivary glands. It is therefore unable to evaluate tumors in the deep lobe of the parotid gland, and minor salivary gland tumors in the oral cavity, pharynx, and tracheo-bronchial tree. −− US does not evaluate deep tissue involvement, perineural spread, bone invasion, and presence of nodes in the oro/ retropharyngeal regions. −− US cannot identify the course of the intraparotid portion of the facial nerve. However, its location can be inferred by identifying the intraparotid portion of the external carotid artery and the retromandibular vein which run alongside the facial nerve. Despite the above limitations, in our experience: US is the ideal initial investigation for: −− Salivary gland mass with no obvious signs and symptoms suggestive of malignancy. −− Masses in the superficial lobe of the parotid gland (where most parotid tumors are located and are benign) and submandibular and sublingual tumors. In this group of patients, the high resolution of US helps to characterize tumors, evaluate associated lymphadenopathy,

Fig.  14.8  Transverse GS US shows a solid, hypoechoic, ill-defined malignant submandibular tumor (arrows). Note the extra-capsular extension into subcutaneous soft tissues (arrowheads)

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Fig. 14.9  Transverse GS US (a) shows typical features of a malignant tumor (arrows). Note its ill-defined edges and heterogenous internal architecture. Corresponding PDS (b) shows marked intratumoral vascularity (arrows)

Fig.  14.10  Transverse GS US shows an ill-defined, heterogeneous parotid mass (arrows) with intratumoral necrosis (arrowheads). The US appearances are suspicious of a malignant tumor, muco-epidermoid carcinoma confirmed at surgery

Fig. 14.11  Transverse GS US shows a well-defined, solid, lobulated, hypoechoic nodule (arrows) with posterior enhancement (arrowheads). The US appearances are typical of a pleomorphic adenoma. Open arrows: mandible

−− Internal architecture: Malignant tumors have a heterogeneous internal architecture with focal areas of hemorrhage and necrosis (Fig.  14.10). Benign tumors such as pleomorphic adenomas (Fig. 14.11) tend to be more homogeneous and show posterior enhancement, whereas Warthin’s tumor (Fig.  14.12) may be heterogeneous with areas of septation and cystic change. Large (>3 cm) pleomorphic adenomas may also demonstrate hemorrhage and cystic change. The presence of calcification (Fig. 14.13) within benign mixed tumors indicates chronicity of the lesion.

9.5% of malignant transformations are seen in patients where the tumor has been present over 15 years [40]. −− Tumor extent: Malignant tumors may be associated with extraglandular spread and invasion of the overlying muscle, subcutaneous tissues, and skin. −− Tumor vascularity (Fig.  14.9b): Malignant tumors are more vascular with a resistive index (RI) > 0.8, pulsatility index (PI) > 1.8 [41], and may demonstrate a hilar vascular pattern compared to pleomorphic adenoma which have peripheral vascularity [42].

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However one must note that: −− Although US may help to differentiate benign from malignant lesions it is unable to distinguish between the various types of malignant tumors. −− Sonographic appearances of low-grade malignant tumors simulate benign salivary gland lesions and guided FNAC may be indicated for some benign looking salivary tumors to rule out a low-grade carcinoma.

Role of Ultrasound in Recurrent Salivary Gland Tumors Distortion of anatomy and scarring often makes ultrasound difficult in the postoperative state. However, due to the superficial location of these recurrent lesions US again is an ideal investigation, and when combined with FNAC it provides the information necessary for treatment planning. Fig.  14.12  Transverse GS US shows a predominantly cystic tumor (open arrow) with internal septa (arrowheads) and a “solid” component (arrows) in the superficial parotid. A similar smaller tumor was seen in the contralateral parotid. FNAC confirmed Warthins tumor

Benign tumors: Pleomorphic adenomas may recur following surgery with a recurrence rate between 1 and 50% [43]. The recurrences are frequently localized to the site of surgery and may be multiple. US readily evaluates these tumors, and the recurrent “nodules” are well-defined, homogeneous with posterior enhancement and peripheral vascularity. Malignant tumors: MR is the investigation of choice for evaluating recurrent disease as in such cases the previous surgery may have been extensive with significant distortion of anatomy. MR clearly evaluates the operative site and extent of invasion of any recurrent tumor. The role of ultrasound is often restricted to guiding a confirmatory biopsy.

Role of Ultrasound in Neck Node Evaluation

Fig.  14.13  Longitudinal GS US shows a well-defined, solid, hypoechoic, parotid pleomorphic adenoma (arrows). Note intratumoral calcification (arrowheads), suggesting a long-standing lesion. Open arrows: mandible

−− Lymphadenopathy: The presence of associated malignant looking nodes in the known draining sites of salivary gland cancer is another clue toward the malignant nature of a salivary mass.

The presence of metastatic nodes in the neck in a patient with HN cancer affects prognosis and treatment options [44, 45]. High-resolution ultrasound, with its excellent spatial resolution, ease of dynamic multiplanar imaging, wide availability, and lack or ionizing radiation, is a recognized modality for the assessment of cervical lymph node metastasis [46–48]. It is superior to CT and MR in its resolution, ability to show vascular characteristics, and the ease to combine with FNAC. The use of Doppler has clearly improved the specificity of ultrasound [49–51] and US + FNAC has a sensitivity of 97% and specificity of 93% [52]. In routine clinical ultrasound of neck nodes, the sonographic features assessed are divided into gray scale features and Doppler parameters. The gray scale features include nodal size, shape, border, internal architecture (echogenicity, nodal hilus, calcification, intranodal necrosis, and intranodal

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reticulation), nodal matting, and associated soft tissue edema. The Doppler parameters include the presence and distribution of intranodal vessels and intranodal vascular resistance. −−

Sonographic Features (Gray Scale and Doppler) that Help to Differentiate Malignant from Benign Nodes −− Size: Nodal size alone cannot differentiate malignant from benign nodes. Nodal size is relevant in (a) increase in nodal size on serial examination in a patient with known HN carcinoma is suspicious for metastasis and (b) serial reduction in size is useful in evaluating patients response to treatment [53]. −− Shape (Figs. 14.14 and 14.15): Normal/benign nodes are elliptical whereas metastatic nodes tend to be round [45, 46, 54]. Similarly, eccentric cortical hypertrophy (due to focal tumor infiltration) is another useful sign to identify nodal metastasis [46]. −− Nodal border (Figs.  14.14–14.16): Malignant nodes are associated with sharp borders, whereas benign nodes have unsharp borders [55]. However, ill-defined border in a metastatic node indicates extracapsular spread [56]. Nodes that have previously received radiotherapy may also have ill-defined borders (Fig. 14.17). −− Echogenicity: Metastatic nodes are usually hypoechoic in relation to adjacent muscle [47, 54] except metastatic nodes from papillary thyroid carcinoma which are often hyperechoic relevant to muscle [32]. −− Nodal Hilus: In a normal neck, most nodes >5 mm will demonstrate the presence of an echogenic hilus [57]. The presence of such an echogenic hilus (Fig.  14.14a) was

Fig. 14.14  Longitudinal GS US (a) shows a elliptical hypoechoic reactive lymph node (arrows). Note the ill-defined border (open arrows) and the linear echogenic hilus (arrowheads). Corresponding PDS (b) shows prominent hilar vascularity (arrowheads)

−−

−−

−−

−−

thought to indicate benignity [45]. However, other studies have shown that the echogenic hilus may also be seen in metastatic nodes [58]. Calcification (Fig.  14.18): Metastatic nodes from papillary carcinoma tend to show punctuate calcification, with faint shadowing on high-resolution ultrasound [32]. Calcification is also seen in a small proportion of metastatic nodes from medullary carcinoma, and nodes treated with chemotherapy or radiotherapy [32, 59]. Intranodal necrosis (Fig.  14.19): Irrespective of nodal size the presence of intranodal necrosis indicates abnormality [59]. It is seen in metastatic and tuberculous nodes in the neck [54, 59]. Intranodal reticulation (Fig.  14.20): It was previously reported that lymphomatous nodes have a pseudocystic appearance, i.e., solid, hypoechoic with posterior enhancement, especially in non-Hodgkin’s lymphoma [60, 61]. However, with the advent of newer high-resolution ultrasound this pseudocystic appearance in non-Hodgkin’s lymphoma is not often seen and an intranodal micronodular reticulated pattern is ­commonly found in lymphomatous nodes [62]. Nodal matting and soft tissue edema: These are commonly seen in tuberculous neck nodes [54]. However, these features may also been seen in metastatic nodes with adjacent soft tissue infiltration and in patients who have received radiation therapy of the neck [63, 64]. Intranodal vascular distribution: Evaluation of the vascular pattern within nodes is a reliable predictor of abnormality [65]. On Doppler, most normal nodes >5 mm will demonstrate the presence of hilar vascularity [57]. Normal or reactive nodes may be apparently avascular or demonstrate hilar vessels (Fig.  14.14b) [49, 66, 67]. However, metastatic nodes demonstrate peripheral or hilar and peripheral (mixed) vascularity (Figs. 14.16, 14.18b, and 14.21)

14  Ultrasound Investigations in Head and Neck Cancer Patients

Fig. 14.15  Transverse GS US shows multiple, solid, hypoechoic metastatic nodes (arrows). Note their sharp borders (arrowheads) and the absence of the echogenic hilus

229

Fig. 14.17  Longitudinal GS US of a metastatic node (arrows) previously treated with radiotherapy. Note the diffuse, ill-defined borders (arrowheads) of the lymph node. The presence of such ill-defined borders in a metastatic node with no previous history of radiotherapy would indicate extracapsular spread

vessels in the node. This is measured as resistive index (RI) and pulsatility index (PI). However, in routine clinical practice such measurements take a lot of time (guided FNAC is much quicker) and their overall values in differentiating benign from malignant nodes is unclear. In our experience, the optimum cut off values for RI and PI are 0.7 and 1.4 with a sensitivity of 86 and 80% and specificity of 70 and 86% respectively [69].

Fig. 14.16  PDS of a metastatic node shows abnormal peripheral vascularity (arrows). Note the ill-defined edges anteriorly (arrowheads) suggesting extracapsular spread

[68, 69]. Therefore, the presence of peripheral intranodal vessels should strongly raise the possibility of metastasis in a patient with known HN carcinoma. This abnormal vascularity is related to angiogenesis within metastatic nodes [66]. −− Intranodal vascular resistance: Using spectral Doppler one can estimate intravascular resistance within small

In addition to the above criteria, the number of nodes in the known draining site of the tumor may also help in predicting their nature. It has been suggested that one should have a high degree of suspicion if there are >3 equivocal/ suspicious nodes in the draining site of the tumor, with specific measurements for minimal axial diameter of the nodes at these sites [70, 71]. One must note that for sonographic evaluation of neck nodes the operator must be familiar with anatomy and pay meticulous attention to detail as many of the nodes and vessels are small. None of the criterion used alone may accurately reflect the nature of the node, and it is a combination of sonographic features that helps in predicting the pathology. In clinical practice, the easiest criteria to evaluate are nodal shape, intranodal necrosis, presence/absence of echogenic hilum, punctuate calcification, and abnormal vascularity. These signs in summation are fairly accurate in predicting the nature of the node and at the same time repeatable and not time-consuming.

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Fig. 14.18  Transverse GS US (a) shows a small, hyperechoic, solid node (arrowheads) with focal punctate calcification (arrow); adjacent to the common carotid artery (open arrow). The sonographic appearances are typical for metastatic lymph node from papillary carcinoma. Corresponding PDS (b) shows profuse, abnormal peripheral vascularity (arrows); typical of a metastatic node

Fig. 14.19  Transverse GS US shows multiple, round metastatic nodes (arrows) from HN SCCa. Note the cystic change (arrowheads) within the nodes. Cystic change within a node, irrespective of nodal size ­indicates abnormality

Fig. 14.20  Transverse GS US shows a lymphomatous (arrow) node with a typical reticulated/micronodular echopattern. Arrowhead identifies CCA and open arrow the IJV

Contrast-Enhanced Ultrasound of Lymph Nodes

parenchymal perfusion [73]. We have used contrast to evaluate treatment response to patients with lymphoma, and showed a delay to peak enhancement ­following treatment [74, 75]. However, the change in the magnitude of peak enhancement was variable after ­treatment with some posttreatment nodes enhancing more than the others. Its use in routine clinical practice is still under consideration.

Contrast-enhanced ultrasound demonstrates more ­intranodal vessels allowing for better visualization and characterization of these vessels [72, 73]. In addition, it provides an objective time-dependent enhancement curves which help to identify the nature of the nodes and better evaluate nodal

14  Ultrasound Investigations in Head and Neck Cancer Patients

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References

Fig. 14.21  PDS of a metastatic node shows abnormal mixed intranodal vascularity, hilar (arrowhead ) and peripheral (arrows)

Role of Ultrasound in Evaluating Posttreatment Nodes Following chemotherapy or radiation therapy it may not always be possible to predict the nature of residual nodes using US. However, in our experience some features that predict good response to treatment are: −− Serial reduction in size of node on treatment −− Serial change in shape of node from round to elliptical −− Reappearance of the echogenic hilum in nodes with absent hilus prior to treatment −− Prompt reduction in intranodal vascularity [76]

Conclusion Despite its limitation in assessing deep seated lesions, ultrasound, combined with FNAC plays an important role in imaging patients with thyroid, salivary gland cancer, and metastatic neck nodes. It is quick, noninvasive, office-based procedure (with a short learning curve), and provides the ­clinician with key information (diagnosis, extent of local and distant disease [77]) necessary to comprehensively manage a patient with head and neck cancer.

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14  Ultrasound Investigations in Head and Neck Cancer Patients 69. Ahuja AT, Ying M, Ho SS, Metreweli C. Distribution of intranodal vessels in differentiating benign from metastatic neck nodes. Clin Radiol. 2001;56:197–201. 70. van den Brekel MW, Stel HV, Castelijns JA, Croll GJ, Snow GB. Lymph node staging in patients with clinically negative neck examinations by ultrasound and ultrasound-guided aspiration cytology. Am J Surg. 1991;162:362–6. 71. van den Brekel MW, Castelijns JA, Stel HV, et al. Occult metastatic neck disease: detection with US and US-guided fine-needle aspiration cytology. Radiology. 1991;180:457–61. 72. Moritz JD, Ludwig A, Oestmann JW. Contrast-enhanced color Doppler sonography for evaluation of enlarged cervical lymph nodes in head and neck tumors. AJR Am J Roentgenol. 2000;174:1279–84. 73. Rubaltelli L, Khadivi Y, Tregnaghi A, et al. Evaluation of lymph node perfusion using continuous mode harmonic ultrasonography

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Chapter 15

Functional Imaging Ludwig G. Strauss and Antonia Dimitrakopoulou-Strauss

Abstract  Functional imaging has found widespread use within the last years due to the increasing availability of PET and PET/CT systems worldwide. The most frequently used tracer for PET examinations is F-18-deoxyglucose, which is transported like glucose into the cells and phosphorylated, but then trapped. Dynamic PET examinations provide detailed information about the kinetics of the tracer and facilitate the detection and delineation of lesions by parametric imaging techniques. However, even static images 1 h after tracer injection are helpful to assess malignant lesions. Tumor diagnostics is improved by the combination of PET and CT or MRI by the use of image fusion techniques. Radiotherapy planning is improved by the inclusion of functional images and the better delineation of a tumor. Other tracers like F-18-FLT provide information about the proliferation, and tracers like F-18-misonidazole can be used to assess hypoxia in tumors. Especially, hypoxia imaging is helpful for the radiotherapy of patients. Keywords  Positron emission tomography • PET • FDG • FLT • Misonidazole • Staging • Therapy management • Parametric imaging

Tumor Diagnostics Morphological Imaging Tumors of the oral cavity and oropharynx are within the most common cancer types in Germany. Like in other tumors, accurate methods are needed for diagnostics and staging to guide the individual patient to the appropriate therapy. Besides clinical methods including endoscopy and the histological assessment of suspicious lesions, morphological

L.G. Strauss (*) CCU Nuclear Medicine, German Cancer Research Center, Im Neuenheimer Feld 280, BW 69120, Heidelberg, Germany e-mail: [email protected]

methods are applied to assess a mass according to size, location, and infiltration of the surrounding region. Furthermore, the detection of metastases is important for staging and therapy planning. Within the last 10 years, MRI has gained increasing interest due to the possibility to obtain noninvasively high contrast images of morphological structures. Leslie et al. evaluated CT and MRI for T- and N-staging of squamous cell carcinoma of the oral cavity and oropharynx in patients with primary or recurrent disease [1]. Interestingly, the accuracy for the staging of primary tumors was 77% for MRI and 67% for CT. In contrast to the staging results, the detection of recurrent tumors was improved with an accuracy of 89% for MRI and 100% for CT [1]. The main problem with both imaging modalities was the N-staging, because the procedures failed to identify small lymph node metastases. Furthermore, changes in cell function may not necessarily be detected with morphological methods alone. The data demonstrate that other methods are needed to improve the staging accuracy, especially for N-staging.

Functional Imaging Functional methods are primarily based on nuclear medicine procedures. Basically, radiopharmaceutical is used to generate functional images. A radiopharmaceutical is generally built from two major parts: the isotope and the pharmaceutical. The isotope is needed to obtain a signal outside the body. Labeling with a positron emitting isotope facilitates imaging with positron emission tomography (PET). Based on the physical properties of positron emitting isotopes, two high energy gamma rays are emitted during decay (511 keV) in an angle of about 180°. This enables the so-called coincidence imaging, which is the base for the superior resolution of PET as compared to conventional nuclear medicine methods. The isotope is linked to a pharmaceutical, which determines what functional information is achieved with an examination. Most of the current PET systems are actually PET/CT systems, which combine the advantages of functional PET imaging with the morphological information obtained by CT.

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_15, © Springer Science+Business Media, LLC 2011

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PET: F-18-Deoxyglucose One of the most common radiopharmaceuticals for PET is F-18-labeled deoxyglucose (FDG), which has found widespread use for oncological studies [2]. FDG is transported like glucose via the glucose transporters into the cells, also phosphorylated by the hexokinases, but then not further metabolized. In contrast to glucose, FDG is usually not a substrate for the sodium-dependent transport system. The dephosphorylation rate of the metabolized FDG is generally low in most of the malignant tumors for at least 1 h. Most of the PET examinations are done as static studies. For this purpose, images are acquired usually 1 h after tracer injection. In contrast to this protocol, at our center we prefer to combine dynamic and static imaging. Therefore, first a 60-min dynamic PET (dPET) study is acquired for the target region, usually the head and neck area. Then after the dPET acquisition additional static images are acquired to obtain partial or whole body images. Based on the dPET acquisition very accurate quantitative data can be obtained about the kinetics of FDG. For quantification purposes we introduced the term “standardized uptake value” (SUV) [2]. The SUV helps to compare different patient studies and follow-up examinations by standardizing the tracer concentration for the injected dose and body weight [2]. The SUV has no dimension and reflects the relative distribution of the tracer in the body. If the tracer would be equally distributed in the whole body an SUV of one would be measured. Based on the FDG transport and phosphorylation in tissue the uptake is usually greater than one. If dPET studies are used, compartment and noncompartment models can be applied to the data to gain detailed information about the tracer kinetics. One of the initial PET studies was done from Minn et al., who compared the FDG data with flow cytometry in head and neck tumors [3]. The authors found no correlation with the histologic grade of the tumors, but a correlation was noted for the FDG uptake ratio and the proliferating cells as measured by flow cytometry [3]. We compared the tumor perfusion using O-15-water and FDG uptake with flow cytometry data in 35 patients with head and neck tumors [4]. Interestingly, the tissue perfusion data did not correlate neither to the FDG uptake nor to the flow cytometry results. The FDG data revealed two subgroups with a significant correlation of the FDG SUVs in each subgroup with the proliferative index [4]. The data demonstrate that functional methods such as PET FDG are closely related to molecular biological processes and may therefore be helpful to assess a malignant lesion in more detail. Several studies have focused on the aspect of diagnostic accuracy of PET in head and neck tumors. Gambhir et  al. performed a meta-analysis of data about the use of PET in oncology [5]. Their evaluation of studies concerning head and neck tumors comprise 298 patients and 580 lesions

L.G. Strauss and A. Dimitrakopoulou-Strauss

assessed for tumor diagnostics and 591 patients with 2,113 lesions evaluated for tumor staging [5]. PET and CT results were compared and an average sensitivity and specificity of 93% and 70% for PET and 66% and 56% for CT were calculated from the literature data for primary tumor diagnostics, respectively. The data were comparable for tumor staging except for a higher specificity of PET and CT as compared to the diagnostic studies (PET: sensitivity 87%, specificity 89%; CT: sensitivity 62%, specificity 72%). The clinical situation, however, is usually associated with a certain prevalence of disease prior to any diagnostic procedure. Diagnostic methods are then applied and it is usually expected that the gain in information enhances the probability or absence of a disease. This is the application of the Bayesian statistics [6]. The data from Gambhir et  al. can be analyzed using the Bayesian approach. Overall, PET provides a higher gain in information as compared to CT. Especially, the rate of falsenegative results with PET is significantly lower as compared to CT. The advantage of combining a morphological method with a functional procedure is also assessed using the literature data and the predicted gain in information is calculated. Especially in patients with a low prior probability of disease the combination of PET and CT will be helpful. This directs to the preferential use of both, morphological and functional methods to achieve the most accurate tumor diagnosis and staging information in individual patients.

Hybrid Systems: PET/CT The recent development of hybrid systems, combining PET and CT, is a major step forward to achieve the most accurate correlation between morphology and function. Due to the sequential acquisition of CT and PET data the misalignment between CT and PET is kept to a minimum and the images can be reviewed side by side or as fusion images. The combined assessment of both, CT and PET, as well as the fusion of images are especially helpful to delineate and locate the viable tumor tissue most accurately. Branstetter et al. compared PET/CT with PET and CT as individual modalities in patients with head and neck tumors [7]. Again, the lowest accuracy was noted for CT (74%), while PET (90%) and PET/CT (94%) were significantly more accurate. When Bayesian statistics is applied to the data, the highest gain in information is obtained with PET/CT. The advantages of PET/CT are also reported by Goerres et al. [8]. The authors are among the first who have used PET/CT in patients and they emphasize that PET and CT are matching with a few millimeters difference. Therefore, the combined use of the imaging modalities improves the presurgical staging by providing both, the anatomical location based on CT and the high lesion detectability of PET with FDG.

15  Functional Imaging

FDG: Sources of Error The metabolically active tracer FDG provides generally a high sensitivity for the detection of abnormalities. Therefore, generally false-negative results are less likely than falsepositive results when FDG is used for tumor diagnostics. However, little is reported about false-positive results. Ng et  al. assessed the clinical usefulness of FDG PET in recurrent nasopharyngeal carcinomas and compared the results with MRI [9]. The overall sensitivity of PET, comprising the primary tumor site and the lymph node metastases, was 89.5%, while the specificity was only 55.6% [9]. One limitation of PET with FDG was the number of falsepositive results obtained in these patients. However, in this study the patients have had received radiotherapy and also chemotherapy had been given to most of the patients. Therefore, there may be a higher likelihood for an enhanced, reactive metabolic activity in tissue according to treatment reflected by an enhanced FDG accumulation.

PET: Non-FDG Tracers The problem of FDG uptake in both, malignant tumors as well as in inflammatory tissue, is already known for PET. False-positive results are mainly based on the enhanced FDG transport into leucocytes. To limit false-positive results, generally two approaches are possible: the use of more sophisticated quantification methods to assess the FDG kinetics in a target volume in detail, or the application of a second radiopharmaceutical to achieve additional biological information

Fig. 15.1  Left: FDG image 60 min after tracer application in a patient with a malignant histiocytoma. Low FDG accumulation due to a low tumor metabolism. Middle: Image  60  min following FLT injection.

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about the lesions. Besides tumor metabolism the assessment of tumor proliferation is another important topic in oncology. The first tracer used to assess tumor proliferation noninvasively with PET was C-11-labeled thymidine. Shields et al. used C-11-thymidine and kinetic modeling to quantify the DNA synthesis rate [10]. In the following years, C-11thymidine was replaced by 3¢-deoxy-3¢-18F-fluorothymidine (FLT). This tracer is also a substrate for the thymidine kinase and associated with the proliferation rate of tumors. In contrast to thymidine, FLT is not incorporated into the DNA, it can only act as a chain terminator. Initial results demonstrate that FLT has limited accuracy regarding the differentiation between reactive and metastatic lymph node metastases [11]. One reason discussed by the authors may be the B-lymphocyte proliferation in reactive lymph nodes. Another reason may be the dependency of FLT kinetics on the extracellular ATP concentration, which has an impact on the structure and performance of the thymidine kinase. Primary tumors usually demonstrate an increased FLT uptake. In some cases, the uptake of FLT may be significantly higher as compared to FDG (Fig. 15.1). Another new tracer is 18F-Galacto-RGD, which binds preferentially to the aVb3 receptor [12]. Integrins are an important group of genes related to tumor growth, invasiveness, and likelihood of metastases. Initial studies suggest the use of these tracers in patients with head and neck tumors, however, further studies must be performed to assess the value of this tracer for the diagnostics or therapy management. Other tracers like F-18-labeled tyrosine or the SSTR2-binding Ga-68-DOTATOC have found limited use. Boer et al. report about a high accuracy of dynamic F-18tyrosine examinations for the detection of recurrent laryngeal tumors [13].

Preferential accumulation of the tracer in the tumor, indicating proliferative activity in the lesion. Right: Fusion image of FDG and FLT

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Therapy Management Diagnostics of Tumor Recurrence One major aim of new diagnostic methods is besides improvements of tumor diagnostics the individualization and optimization of therapy. PET with FDG is an established procedure for the follow-up of patients for tumor recurrence since several years now [2]. In head and neck tumors, one important topic for follow-up is the improved detection of a recurrent tumor. Based on the data from Gambhir et al., who included 426 patients in the meta-analysis, the sensitivity of FDG PET on a patient-based analysis is 93% and the specificity is 83% (CT: 54% sensitivity, 74% specificity) for the detection of recurrent head and neck tumors [5]. These data are comparable to those obtained for tumor diagnostics. PET FDG studies are usually performed for therapy monitoring to assess changes in tumor metabolism following treatment. The evaluation of 169 patients demonstrated a sensitivity of 84% and specificity of 95% for the assessment of therapyrelated changes in the tumor [5]. Again, PET was superior to CT (60% sensitivity, 39% specificity), because usually functional changes precede changes in tumor volume. Gambhir et al. noted a correlation of tracer kinetics and growth rates and report about a 33% change in treatment management due to PET results [5]. The combination of PET and CT is currently the state-of-the-art technique. The PET/CT systems used for FDG studies in patients with head and neck cancer provide easy access to both morphology and function. Some systems provide the possibility to perform the scans with localizer devices for radiotherapy. We use, additionally,

L.G. Strauss and A. Dimitrakopoulou-Strauss

parametric images of the global metabolic rate and also fusion of the parametric images with CT and MRI in patients with recurrent head and neck cancer (Fig. 15.2).

Prognostic Aspects of PET The prognostic value of PET with FDG was investigated in a few studies. Based on the results obtained by Minn et al. [3] and Haberkorn et  al. [4], it can be expected that the quantitative evaluation of the FDG uptake may be helpful to assess the proliferative aspect of tumors. Halfpenny et al. evaluated the association of FDG uptake, as measured by the SUV, and therapy outcome [14]. The authors performed FDG studies in 73 patients prior to treatment (surgery and radiotherapy). An SUV of 10 was used for Kaplan Meier analysis and revealed a highly significant difference in survival [14]. Therefore, the quantitative data of the initial FDG uptake prior to treatment are predictive for therapy outcome. We investigated the correlation of changes in FDG uptake and tumor growth rates in patients with head and neck tumors, receiving a cisplatinum-based chemotherapy [15]. Dynamic PET studies were performed prior and after one chemotherapeutic cycle, the changes in tracer uptake were compared to the changes in tumor volume. The growth rates and the changes in FDG uptake (SUV) were correlated for both tumors (r = 0.98) and lymph node metastases (r = 0.94). Interestingly, the growth rates were different for the same changes in the FDG uptake. Overall, tumors were more sensitive to treatment than lymph node metastases [15].

Fig. 15.2  CT (left) and dPET study with FDG (middle) of a patient with a recurrent squamous cell carcinoma. Parametric images of the FDG metabolic rate were calculated from the dPET data and fused with CT (right)

15  Functional Imaging

Assessment of Hypoxia One problem in patients undergoing radiotherapy is the impact of hypoxia on treatment. Tumor resistance can be induced, e.g., by the hypoxia-inducible factor a (HIF-1a). Furthermore, the von Hippel–Lindau (VHL) gene is involved in the mechanisms of hypoxia, angiogenesis, and radioresistance. Several studies have shown that these genes are important for the biological effects of radiotherapy and that the inhibition, e.g., of HIF-1a may result in an either enhanced or decreased therapeutic effect, dependent on the timing when the inhibitor is applied during radiation therapy [16, 17]. Hypoxia is closely related to other biological factors such as angiogenesis, metabolism, and proliferation. Interestingly, even hypoxia and FDG metabolism are not independent parameters and hypoxia is affecting glucose metabolism. We noted an association of HIF-1a expression and the pharmacokinetics of FDG in colorectal tumors [18]. In particular, the expression of HIF-1a correlated with the SUV, FDG influx (global metabolic rate), k3 (intracellular phosphorylation), and the fractal dimension (heterogeneity) of the tracer kinetics. In giant cell tumors, a correlation of HIF-1a and k3 was found, giving evidence for a modulation of the FDG kinetics by hypoxia-related gene expression [19]. The results direct to an impact of hypoxia on the FDG kinetics. If the impact of hypoxia on the FDG kinetics is known, the amount of hypoxia may be predicted from the dPET data using dedicated regression functions. However, data are reported only for colorectal tumors and giant cell tumors, results are missing for head and neck tumors. The best approach to assess hypoxia in tumors is to use a tracer with close dependency on tissue oxygen pressure. Several approaches were already made to use a hypoxiaspecific tracer for PET studies. One of the most common radiopharmaceuticals for hypoxia imaging is F-18fluoromisonidazole (FMISO), which accumulates in tissues with oxygen concentrations of less than 3  mmHg. Studies have shown that FMISO can be used to detect and quantify local tissue hypoxia. One of the first reports is a study from Mathias et al. about the use of F-18-Misonidazole for the imaging of hypoxia in ischemic myocardium and brain [20]. One limitation of FMISO is the slow pharmacokinetics, demanding imaging either dynamically for more than 1 h or late images at least 90 min after tracer application. Several studies were performed in different tumor types. Koh et al. used F-18-misonidazole in patients with locally advanced nonsmall cell lung cancer prior to radiotherapy [21]. Furthermore, in some patients early treatment follow-up studies were also performed. The results gave evidence for a general tendency toward improved oxygenation during radiotherapy. However, patients with more radioresistant hypoxic tumors could be selected by a single pretreatment evaluation with PET and F-18-misonidazole [21]. Tumor hypoxia was

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evaluated in head and neck tumors by Rajendran et al. in 73 patients prior to treatment and they compared the PET results to therapy outcome [22]. The tumor-to-blood ratios were helpful for the classification of the patients according to overall survival using a cut off value of 1.5: lower ratios were associated with a longer overall survival [22]. The range of the ratios was 1.0–3.5, reflecting a moderate retention of the tracer in hypoxic tumors. However, a more delayed imaging protocol may be helpful in order to achieve higher ratios. One limitation of F-18-misonidazole is the long delay for tumor imaging of 90–120  min following tracer injection. Therefore, other tracers were evaluated as hypoxic markers. Besides FMISO, nitroimidazole analogs and tracers based on thiosemicarbazones (e.g., Cu-60-ATSM) have found increasing attention due to the shorter time interval required for the assessment of tissue hypoxia. Postema et  al. used the substance 1-alpha-d: -(5-deoxy-5-[(18)F]fluoroarabinofuranosyl)-2-nitroimidazole (F-18-FAZA) as a hypoxic marker in 50 patients with head and neck cancer, small cell as well as nonsmall cell lung cancer, malignant lymphoma, and high-grade gliomas [23]. The uptake ratios ranged from 1.9 to 15.6 and were higher as compared to the results reported for F-18-misonidazole. However, the delay needed for scanning was comparable for both tracers, therefore the major advantage of F-18-FAZA are the higher retention ratios as compared to F-18-misonidazole. Other tracers have been investigated in order to shorten the delay time required for imaging. Holland et  al. report in a detailed review about the properties of one of the most promising new tracers, the copper(II) complex of diacetyl-2,3bis(N4-methyl-3-thiosemicarbazonato) ligand (Cu-ATSM) [24]. ATSM can be labeled with Cu-60 or Cu-64 and is useful for PET. Cu-ATSM is trapped in hypoxic cells and reflects the amount of hypoxia in tissue. In most studies, a delay of about 30  min following tracer injection is used for PET imaging [24]. Kinetic studies demonstrated that even 10  min after tracer application, constant activity levels may be achieved. Cu-60 labeling has the advantage of a short half-life (t1/2 = 32.7  min) and is therefore helpful to perform double tracer studies. Cu-64 (t1/2 = 12.7 h) may be helpful to achieve a somewhat higher contrast. Dence et  al. compared F-18misonidazole, Cu-64-ATSM, FDG, and FLT in a rodent model of cancer [25]. The results demonstrated an excellent correlation for F-18-misonidazole and Cu-64-ATSM, either at 10  min postinjection (R2 = 0.84) or 24  h (R2 = 0.86) [25]. Interestingly, also a significant correlation was noted for Cu-64-ATSM and FLT (R2 = 0.829), directing to an association of hypoxia and tumor proliferation rate. The current results show that FDG is helpful to achieve functional data about a tumor in the head and neck region. Dependent on the planned treatment, other tracers may be used, e.g., to detect and quantify hypoxia in tumors, which is important for radiotherapy and provides prognostic information.

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References 1. Leslie A, Fyfe E, Guest P, Goddard P, Kabala JE. Staging of squamous cell carcinoma of the oral cavity and oropharynx: a comparison of MRI and CT in T- and N-staging. J Comput Assist Tomogr. 1999;23:43–9. 2. Strauss LG, Conti PS. The applications of PET in clinical oncology. J Nucl Med. 1991;32:623–48. discussion 649–50. 3. Minn H, Joensuu H, Ahonen A, Klemi P. Fluorodeoxyglucose imaging: a method to assess the proliferative activity of human cancer in vivo comparison with DNA flow cytometry in head and neck tumors. Cancer. 1988;61:1776–81. 4. Haberkorn U, Strauss LG, Reisser C, et al. Glucose uptake, perfusion, and cell proliferation in head and neck tumors: relation of positron emission tomography to flow cytometry. J Nucl Med. 1991;32:1548–55. 5. Gambhir SS, Czernin J, Schwimmer J, Silverman DH, Coleman RE, Phelps ME. A tabulated summary of the FDG PET literature. J Nucl Med. 2001;42:1S–93S. 6. Strauss L, Bostel F, Clorius JH, Raptou E, Wellman H, Georgi P. Single-photon emission computed tomography (SPECT) for assessment of hepatic lesions. J Nucl Med. 1982;23:1059–65. 7. Branstetter BFt, Blodgett TM, Zimmer LA, et  al. Head and neck malignancy: is PET/CT more accurate than PET or CT alone? Radiology. 2005;235:580–6. 8. Goerres GW, von Schulthess GK, Steinert HC. Why most PET of lung and head-and-neck cancer will be PET/CT. J Nucl Med. 2004;45 Suppl 1:66S–71S. 9. Ng SH, Joseph CT, Chan SC, et al. Clinical usefulness of 18F-FDG PET in nasopharyngeal carcinoma patients with questionable MRI findings for recurrence. J Nucl Med. 2004;45:1669–76. 10. Shields AF, Lim K, Grierson J, Link J, Krohn KA. Utilization of labeled thymidine in DNA synthesis: studies for PET. J Nucl Med. 1990;31:337–42. 11. Troost EG, Vogel WV, Merkx MA, et al. 18F-FLT PET does not discriminate between reactive and metastatic lymph nodes in primary head and neck cancer patients. J Nucl Med. 2007;48:726–35. 12. Beer AJ, Haubner R, Wolf I, et al. PET-based human dosimetry of 18F-galacto-RGD, a new radiotracer for imaging alpha v beta3 expression. J Nucl Med. 2006;47:763–9.

L.G. Strauss and A. Dimitrakopoulou-Strauss 13. De Boer JR, Pruim J, Burlage F, et al. Therapy evaluation of laryngeal carcinomas by tyrosine-pet. Head Neck. 2003;25:634–44. 14. Halfpenny W, Hain SF, Biassoni L, Maisey MN, Sherman JA, McGurk M. FDG-PET. A possible prognostic factor in head and neck cancer. Br J Cancer. 2002;86:512–6. 15. Haberkorn U, Strauss LG, Dimitrakopoulou A, et  al. Fluorodeoxyglucose imaging of advanced head and neck cancer after chemotherapy. J Nucl Med. 1993;34:12–7. 16. Harada H, Kizaka-Kondoh S, Li G, et  al. Significance of HIF-1active cells in angiogenesis and radioresistance. Oncogene. 2007;26:7508–16. 17. Harada H, Itasaka S, Zhu Y, et al. Treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the effect of radiation therapy. Br J Cancer. 2009;100:747–57. 18. Strauss LG, Klippel S, Koczan D, et  al. Correlation of hypoxia associated genes with FDG uptake and kinetics [abstract]. J Nucl Med. 2008;49(Suppl):346P. 19. Strauss LG, Dimitrakopoulou-Strauss A, Koczan D, et al. 18F-FDG kinetics and gene expression in giant cell tumors. J Nucl Med. 2004;45:1528–35. 20. Mathias CJ, Welch MJ, Kilbourn MR, et al. Radiolabeled hypoxic cell sensitizers: tracers for assessment of ischemia. Life Sci. 1987;41:199–206. 21. Koh WJ, Bergman KS, Rasey JS, et al. Evaluation of oxygenation status during fractionated radiotherapy in human nonsmall cell lung cancers using [F-18]fluoromisonidazole positron emission tomography. Int J Radiat Oncol Biol Phys. 1995;33:391–8. 22. Rajendran JG, Schwartz DL, O’Sullivan J, et  al. Tumor hypoxia imaging with [F-18] fluoromisonidazole positron emission tomography in head and neck cancer. Clin Cancer Res. 2006;12:5435–41. 23. Postema EJ, McEwan AJ, Riauka TA, et al. Initial results of hypoxia imaging using 1-alpha-D: -(5-deoxy-5-[(18)F]-fluoroarabinofuranosyl)2-nitroimidazole ( (18)F-FAZA). Eur J Nucl Med Mol Imaging. 2009;36(10):1565–73. 24. Holland JP, Lewis JS, Dehdashti F. Assessing tumor hypoxia by positron emission tomography with Cu-ATSM. Q J Nucl Med Mol Imaging. 2009;53:193–200. 25. Dence CS, Ponde DE, Welch MJ, Lewis JS. Autoradiographic and small-animal PET comparisons between (18)F-FMISO, (18) F-FDG, (18)F-FLT and the hypoxic selective (64)Cu-ATSM in a rodent model of cancer. Nucl Med Biol. 2008;35:713–20.

Chapter 16

Sentinel Node Biopsy for Head and Neck Cancer Lee Alkureishi and Gary L. Ross

Abstract  The presence of cervical lymph node metastases remains one of the most important prognostic factors for various solid tumors of the head and neck, including melanoma, squamous cell carcinoma (SCC), and Merkel cell carcinoma (MCC). In patients with clinically evident neck involvement, the regional lymphatics clearly require directed treatment, and this may involve therapeutic neck dissection or radiotherapy. However, the decision whether or not to electively treat patients with clinically uninvolved cervical lymphatics is usually less clear-cut. On the one hand, elective neck dissection simultaneously allows for accurate pathological neck staging and definitive surgical management of patients found to harbor occult metastatic disease. On the other hand, the majority of patients with clinically negative (cN0) necks do not harbor occult disease and would therefore be overtreated by an elective neck dissection. The significant morbidity associated with neck dissection means that this is a real concern, and efforts to minimize the extent of surgical intervention while maintaining oncologic safety are ongoing. The radical en bloc cervical lymph node dissections introduced at the start of the twentieth century have largely been surpassed by more focused surgical procedures, including the modified radical neck dissection (MRND) and more recently, selective neck dissection (SND). The operative morbidity of MRND and SND procedures compares favorably with more extensive dissections, though it remains significant. Sentinel lymph node biopsy (SLNB) represents an extension of this principle; by super-selecting the small subset of lymph nodes most likely to harbor disease, the extent of surgical intervention can be further minimized without adversely affecting diagnostic accuracy. The sentinel node concept states that tumor spread occurs in a stepwise progression from the primary tumor to the first-echelon lymph nodes, before progression to the remainder of the lymphatic basin.

G.L. Ross (*) Plastic Surgery Department, The Christie, Wilmslow Road, Manchester, M20 4BX, UK e-mail: [email protected]

These first-echelon lymph nodes, known as the sentinel nodes, can be harvested, examined for the presence of tumor, and used to predict the disease status of the entire basin. In the head and neck region, considerable variability exists in the patterns of lymphatic drainage from each primary tumor site, and the exact location of the sentinel nodes therefore varies between patients. In order to accurately locate the SLNs, a number of techniques may be employed. Preoperatively, radio-labeled tracer is injected in a peritumoral fashion, traveling via the lymphatics to the first-echelon nodes, where it may be detected by gamma camera during lymphoscintigraphy (LSG). A handheld gamma probe is utilized intraoperatively to afford more precise radiolocalization, and some surgeons choose also to inject peritumoral blue dye, easing visual identification of the lymphatics. These comprise the sentinel lymph node biopsy technique, which has been applied to a variety of solid tumors, including breast cancer, malignant melanoma (MM), and penile cancer. This chapter describes SLNB as it relates to the management of solid tumors in the head and neck region, particularly malignant melanoma, SCC, and MCC. A brief history of the development of the technique and its reported accuracy are presented, and the advantages and disadvantages of this relatively new application are discussed. Finally, this chapter explores the possible roles that SLNB may play in the future management of head and neck cancer. Keywords  Head and neck cancer • Oral cancer • Squamous cell carcinoma • Sentinel node biopsy

Introduction Head and neck cancers comprise a diverse group of tumors arising from the epidermis, with significant differences in tumor biology, disease characteristics, and prognosis. The three most common types of head and neck cancer are malignant melanoma (MM), arising from melanocytes; squamous cell carcinoma (SCC), arising from keratinocytes;

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_16, © Springer Science+Business Media, LLC 2011

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Fig. 16.1  (a) Individual lymph node groups in the head and neck, grouped into superficial and deep jugular chains. (b) Robbins’ Classification of cervical lymph node levels

and Merkel cell carcinoma (MCC), a rare aggressive skin tumor arising from neuroendocrine cells. Despite their differences in many regards, these cancer types share one important characteristic; their prognosis is heavily dependent on the presence or absence of lymph node metastases. Patients with malignant melanoma and nodal involvement demonstrate less than 50% 5-year survival [1], and similar figures have been reported for patients with SCC [2]. In MCC, the presence of nodal disease has been shown to be the most important prognostic indicator by multivariate analysis [3], with a further study demonstrating a drop from 40 to 13 months median survival with nodal involvement [4]. Virchow [5] was the first to postulate that lymph nodes act as a barrier to particulate matter, and in particular cancer cells. The contention that cancer progression followed a sequential route from the primary site to the regional lymphatics before distant metastasis laid the way for the development of regional surgical treatments for a variety of cancers; first, Halsted radical mastectomy for breast cancer [6]; and in the case of the head and neck, the radical neck dissection (RND) as described by Crile [7].

Anatomy of the Cervical Lymph Node Basin The lymphatic anatomy of the head and neck is complex, comprising approximately 250–350 lymph nodes and demonstrating great variability in the patterns of lymph flow observed [8]. The cervical lymph nodes may be divided into superficial and deep chains. The superficial chain lies between the skin and the superficial fascia of the face and scalp, following the anatomy of the major veins, and eventually

drains into the deep chain. The deep chain lies along the course of the internal jugular vein under the sternocleidomastoid muscle, draining inferiorly from the base of the skull to the brachiocephalic junction, where lymph is returned to the venous system. The most popular system of classification for cervical lymphatic anatomy was developed at the Memorial Sloan-Kettering Cancer Center [9], and forms the basis for describing the various types of neck dissection in current usage [10]. In this system, the cervical lymph nodes are divided into levels I through VI. The anatomy and classification system are illustrated in Fig. 16.1.

Neck Dissection The introduction of the RND in 1906 [7] represented an important step for both staging and treatment of patients with head and neck cancer. However, the morbidity associated with such an extensive dissection was considerable. Complications included shoulder stiffness, pain, muscle atrophy, facial swelling, and cosmetic defects while the mortality rate following bilateral RND was reported as high as 10% [11]. A number of “modified radical” neck dissections were developed as a means of minimizing associated morbidity, being designated MRND I–III depending on the structures preserved (accessory nerve, sternocleidomastoid and/or internal jugular vein) [12]. Studies demonstrating the oncologic safety of the MRND led to its adoption as the standard of care, and the RND fell out of favor [13]. The goal of reducing morbidity continues to push the development of more conservative surgical management techniques, however, and this is particularly true for patients

16  Sentinel Node Biopsy for Head and Neck Cancer

with clinically uninvolved necks. Improved understanding of the lymphatic anatomy of the head and neck has facilitated the development of more selective lymphadenectomies, concentrating on the groups of lymph nodes most likely to be involved [14–16]. These selective neck dissections (SNDs) require less extensive dissection, leaving more of the normal lymphatic anatomy intact and have been shown to cause less morbidity when compared with MRND [17]. The various types of neck dissection are outlined in Table 16.1. Despite these recent advances, neck dissection remains an invasive procedure with appreciable morbidity [18] and, while its use in clinically node-positive patients is well established, elective neck dissection for patients with clinically negative (cN0) necks remains controversial. Traditionally considered the gold standard, END provides tissue for accurate pathologic staging while also treating the neck by removing lymph nodes at risk for involvement [19]. However, the majority of cN0 patients do not in fact harbor occult nodal metastases, and may be unnecessarily subjected to the morbidity associated with the procedure. As a result, selection of patients who would benefit most from neck dissection becomes increasingly important. Clinical staging of the cervical lymph nodes is unreliable, with poor reported sensitivities for both palpation and clinical imaging, and it is generally accepted that an occult nodal metastasis rate of 20–30% persists despite meticulous clinical staging [20–22]. For SCC, elective neck dissection is currently recommended for patients with a greater than 20% risk of occult nodal metastases based on primary tumor characteristics, such as site and T-stage [23]. The role of END for cN0 head and neck melanoma patients is unclear, with no consistent survival benefit demonstrated [24]. It has been suggested that END may be most beneficial for patients with primary tumors between 1.5 and 3.99 mm in thickness [25].

Table 16.1  Neck dissection classification 1991 Classification 2001 Classification 1. Radical neck dissection 1. Radical neck dissection 2. Modified radical neck dissection 2. Modified radical neck dissection 3. Selective neck dissection 3. Selective neck dissection a.  Supraomohyoid Each variation is depicted by “SND” b.  Lateral And the use of parentheses c.  Posterolateral to denote d.  Anterior The levels or sublevels removed 4. Extended neck dissection 4. Extended neck dissection From Robbins KT, Clayman G, Levine PA, Medina J, Sessions R, Shaha A, Som P, Wolf GT. Neck dissection classification update: revisions proposed by the American Head and Neck Society and the American Academy of Otolaryngology-Head and Neck Surgery. Arch Otolaryngol Head Neck Surg. 2002;128(7):751–8. Reprinted with permission. Copyright © 2002 American Medical Association. All rights reserved

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Sentinel Node Biopsy Sentinel node biopsy (SNB) represents a means of superselecting the group of lymph nodes most at risk for disease involvement, allowing histopathologic staging of the neck while minimizing the extent of surgical intervention for patients without nodal involvement. The sentinel node concept is based on the assumption that spread from the primary tumor occurs to a single node (or group of nodes) before progressing to the remaining nodal basin and systemic metastasis (Fig. 16.2). Identification of these sentinel nodes allows for selective biopsy and pathologic evaluation of the nodes most likely to represent the disease status of the remaining nodal basin [26]. The results of SNB can then be used to guide further management, with SNB-positive patients going on to receive definitive (therapeutic) neck dissection and/or parotidectomy while SNB-negative patients may be followed clinically. These SNB-negative patients may therefore avoid some of the morbidity associated with neck dissection [27]. The potential advantages of SNB over neck dissection are many-fold, including its minimally invasive nature, a lower per-patient cost compared with comprehensive neck dissection [28, 29], and a drastic reduction in the number of lymph nodes submitted for pathologic evaluation. In turn, this allows a more in-depth search for micrometastatic deposits utilizing techniques, such as step-serial sectioning (SSS) and immunohistochemistry [30, 31]. However, SNB can be a technically challenging technique with a steep learning curve [26, 32] and as such, investigators wishing to begin using the technique for SCC are recommended to do so within the context of SNB-assisted END [33]. As with any biopsy technique, there exists the potential for sampling error and the reported false-negative rate ranges from 0 to 10.5% in most studies for both SCC and melanoma [33–39]. Finally, the usefulness of SNB is currently restricted to cN0 patients, since distortion of the normal lymphatic anatomy by extensive tumor infiltration may lead to unexpected drainage patterns and increase the likelihood of false-negative results [40].

Development of the Sentinel Node Concept The first description of a “sentinel” lymph node dates back to 1960 with a total parotidectomy reported by Gould et  al., during which frozen section examination of a single facial lymph node was used to guide the decision for neck dissection [41]. Subsequently, Cabanas et al. reported direct drainage from the penis to the lymph nodes associated with the superficial epigastric vein in a series of 46 patients with penile SCC and described 90% survival for sentinel nodenegative patients [42]. Similarly, Weissbach and Boedefeld suggested a limited retroperitoneal lymph node dissection in

244 Fig. 16.2  The sentinel node concept

L. Alkureishi and G.L. Ross x x

x Tumor

x

x x

Lymphatic afferent channel

x

Sentinel lymph node

Regional lymph nodes

x = injection of radiotracer/blue dye = tumor deposits

patients with testicular cancer, in order to detect lymphatic involvement while minimizing operative intervention [43]. Holmes et al. introduced the use of colloidal gold injections to demonstrate the actual patterns of lymph drainage for ambiguous areas, such as the midline [44], and followed this in 1992 with the description of intraoperative vital dye injection, providing a means of visually tracing dye-stained lymphatics to the first-echelon nodes [26]. In 1993, Alex and Krag described the intraoperative use of a handheld gamma probe, easing detection of the sentinel nodes and improving identification rates [45]. Since these early studies, SNB has gone on to become increasingly important as a staging tool for patients with early-stage melanoma [46], and work is underway to fully elucidate its utility in SCC management [33, 47]. The role played by SNB in the management of these and other head and neck cancers is described later in this chapter.

Technique of Sentinel Node Biopsy In general, SNB comprises three parts: preoperative lymphoscintigraphy (LSG), intraoperative identification and harvest, and pathological evaluation of sentinel nodes. These components are described in detail in this section, with reference to the minor differences in protocol for each of the major head and neck cancer types.

Preoperative Lymphoscintigraphy The lymphatic anatomy of the head and neck is complex and variable, with discordance between predicted and actual lymphatic drainage in up to 67% of patients [8]. Aberrant drainage patterns can lead to inaccurate placement of the initial access incision, and may contribute to the failure of sentinel node identification [15]. The goal of preoperative LSG is to demonstrate the location of sentinel nodes prior to incision. This begins with injection of a radio-labeled colloid solution at the site of the primary tumor. The radiocolloid may then track along the same afferent lymphatics draining the tumor, accumulating in the first-echelon lymph nodes where the resultant radioactivity may be detected by gamma camera. LSG may be carried out up to 24 h before surgery, or on the day of surgery, and this should be coordinated between the nuclear medicine physician and the surgeon. The technique of radiocolloid injection varies according to the type of cancer being studied. For melanoma and other cutaneous tumors, multiple intradermal injections should be employed to completely encircle the tumor or site of previous excision biopsy. There has been considerable debate regarding the accuracy of LSG, and SNB in general, in cases where wide local excision (WLE) has previously been carried out. While it is strongly preferred that SNB be performed prior to excision, there is some evidence to suggest that previous WLE is not an absolute contraindication [48]. For intraoral lesions, the majority of which are SCC, multiple mucosal/

16  Sentinel Node Biopsy for Head and Neck Cancer

submucosal injections should be performed around the periphery of the tumor or scar margin, and deeper injections may be employed according to the depth of the lesion [49]. Ideally, the operating surgeon should be present for the injections to ensure consistency with injection of blue dye if used. The volume injected varies according to the location and size of the lesion, and ranges from two to four aliquots. A mouthwash should be employed following intraoral injections, to prevent sumping or swallowing of radiotracer. The ideal radiotracer should emit only gamma rays, be cleared rapidly from the injection site, have a uniform particle size, and should persist in the lymph nodes until imaging can be performed [50, 51]. A variety of Technetium99m (99Tcm)-labeled colloids are available, including 99Tcm human serum albumin, 99Tcm colloidal albumin, 99Tcm antimony sulfur colloid, and 99Tcm sulfur colloid, although regional licensing issues may restrict the available choices. In Europe and parts of the USA, Albures™ and Nanocoll™ (Nycomed Amersham, Buckinghamshire, UK) are the most commonly available colloidal albumin preparations. The larger particle size of Albures™ (500 nm) limits its use to primary tumor sites with high lymphatic density, such as the anterior tongue or floor of the mouth, while the 50 nm particle size of Nanocoll™ allows its use in other sites [33, 51]. For regions where human albumin-based colloids have not been approved, sulfur colloid preparations are available in both unfiltered (300–340 nm) and filtered (< 200 nm) forms [52]. There is little consensus on the optimum activity for injection, which varies from 15 to 120 MBq between studies with higher doses or repeat injections being employed for the 2-day protocol [53–55]. However, it has been suggested that much lower doses (0.37–2.2 MBq) may be used in the setting of head and neck melanoma [56]. Planar lymphoscintigraphic imaging may be static or dynamic, or a combination of the two. The addition of dynamic imaging for melanoma patients improves the detection of “intransit” nodes, which are reported to occur in 5–8% of the population and should also be considered sentinel nodes [57, 58]. To date, there have been no reports of in transit nodes in patients with SCC. There is currently no evidence favoring either technique in these patients, and the exact timing of static image acquisition varies between centers. Images should be obtained in two planes: anterior and lateral or lateral-oblique. A gamma camera fitted with a low energy, high resolution (LEHR) collimator is used to image the patient, whose silhouette can be delineated by a flood source of 57Co or 99mTc placed behind the patient or by tracing his/her outline with a 57 Co-labeled marker pen. At this point, it may be helpful to mark the skin overlying visualized sentinel nodes with indelible marker pen [33, 49, 51]. However, this practice has not been universally accepted due to concerns that the change in positioning between LSG and surgery may misguide the placement of initial access incision [59].

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Recent studies have reported potential improvements in preoperative sentinel node identification through the use of Single Photon Emission Computed Tomography (SPECT/ CT) imaging [60, 61]. This hybrid anatomical/functional imaging modality affords better topographical orientation and separation of SLNs from adjacent structures, compared with planar LSG alone. In the melanoma literature, it appears that SPECT/CT can lead to more accurate incision placement and improvements in SLN detection rates [61, 62]. However, these advantages of SPECT/CT imaging have not been consistently demonstrated in the SCC population [63].

Surgical Technique Within 24  h of LSG, patients may undergo the operative portion of SNB. Although SNB of cervical lymph nodes under local anesthesia has been reported [64], most surgeons prefer to employ general anesthesia for this technique. The patient is prepared and draped as for a standard excision and neck dissection. Preoperative LSG images should be available for reference in the operating suite, in electronic or hard copy form, and these may be used to guide the placement of the initial access incision. If skin markings have been placed in the nuclear medicine suite, underlying radioactivity levels should be verified using a handheld gamma probe prior to making the incision. The orientation of the incision should be such that it may be easily excised in the event of a future neck dissection. If injection of vital (blue) dye is desired, this may be carried out prior to preparing and draping. Injections should be undertaken by the same operator as the radiotracer injection in order to ensure consistency, and the pattern and depth of injection should mirror that of the radiotracer. The brand of dye used varies according to geographical region, with Patent Blue V Dye (Laboratoire Guerbet, Aulnay-Sous-Bois, France) available in Europe and Lymphazurin™ (Tyco Healthcare Group LP, Norwalk, CT, USA) in the USA. The technique of blue dye injection, introduced by Morton et al., provides a means of visually identifying the small lymphatic vessels intraoperatively, allowing them to be traced to the first-echelon nodes [26]. However, the success rate of identification of SLNs by blue dye injection is less than that of radiolocalization by gamma-probe, and the technique has a steeper learning curve [65]. In a study of 55 patients with head and neck melanoma, Wells et al. reported a 67% identification rate by blue-dye mapping and 95% utilizing a combined approach [38]. While most blue dye-stained SLNs are also found to be radioactive or “hot,” a small minority of SLNs are “cold,” and proponents of blue dye injection report the facilitation of intraoperative identification [33, 49, 66]. The major perceived

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disadvantages to blue dye are related to persistent cutaneous staining and masking of true surgical margins; however, rare cases of anaphylactic reactions have also been reported [67]. As a result, the use of blue dye is considered optional, though many authors employ a combined approach. Guided by the preoperative LSG images, skin markings (if present) and the handheld gamma probe, a small skin incision (2–4  cm) is made and limited skin flaps elevated. Dissection is carried through the superficial fascia, and is guided by the handheld gamma probe. If blue-stained lymphatics are visualized, these may be followed to the draining lymph node(s); if no staining is present (or dye was not used), the dissection may be guided solely by the gamma probe, which is fitted with a 14  mm diameter straight collimated probe. The angle of the probe may be gradually altered while watching or listening for a change in the counts-per-second (cps). In cases where the primary tumor site lies in close proximity to the regional lymph nodes, radioactive “shinethrough” from the primary tumor site may mask the true position of the sentinel node. In these patients, the use of malleable lead plates between the injection site and the nodal basin may address this issue [26, 45, 49, 51]. All radioactive and/or blue-stained nodes are clipped and excised, and radioactivity is confirmed ex-vivo. Following excision, the remaining basin is examined with the gamma probe and no further SLNs are considered present when the residual count-rate is less than 10% that of the “hottest” excised SLN [68]. Patients undergoing SNB-assisted END may then proceed to completion neck dissection.

Pathologic Evaluation of Sentinel Nodes Detection of metastatic disease in sentinel nodes by pathologic examination is intrinsic to the success of the procedure, and offers a number of advantages over traditional elective

Table 16.2  Comparison of UICC and TNM classifications of micrometastases and isolated tumor cells

neck dissection. Principally, the absolute number of lymph nodes examined is far fewer during SNB, allowing the pathologist to perform a more thorough search for micrometastatic deposits.

Metastases, Micrometastases, and Isolated Tumor Cells Occult metastases may be defined as those found in patients with cN0 necks, and may be subdivided into metastases (greater than 2  mm), micrometastases (³0.2  mm and £ 2  mm), and isolated tumor cells (ITC; <0.2  mm, single cells or small clusters, with no stromal reaction and no contact with vessel wall) according to the most recent International Union Against Cancer (UICC) classification. The relationship of this classification to the most recent AJCC Tumor-Node-Metastasis (TNM) classification of malignant tumors is illustrated in Table 16.2 [69]. In order to compare results across studies, uniform reporting standards for pathologic staging are critical. When SNB is undertaken, the designation (sn) should be added after the N category. The finding of ITCs does not upstage the cN0 neck, and should be reported as pN0 (i+)(sn) while micrometastatic disease results in upstaging and is reported as pN1 (mi)(sn). For each of the head and neck cancer types, the sequence of pathologic examination is broadly similar, and involves gross examination, bivalving of the lymph node, sectioning at predefined intervals and staining with a variety of histopathologic techniques. However, there are a number of minor differences in protocol according to the type of tumor being studied, and exact sectioning/staining protocols vary between centers. In some cases, additional techniques, such as real-time polymerase chain reaction (RT-PCR), may also be employed; these differences are briefly outlined below [70, 71].

Generic TNM coding for sentinel nodes pNX (sn) Sentinel lymph node could not be assessed pN0 (sn) No sentinel node metastasis pN1 (sn) Sentinel node metastasis Sentinel nodes with micrometastasis only are identified by (mi) pN1 (sn)(mi) Single ipsilateral node with micrometastasis pN2 (sn)(mi) Multiple ipsilateral nodes with micrometastasis SLNs with ITC are coded separately for morphological and nonmorphological techniques pN0 (i−)(sn) No SLN metastasis histologically, negative morphological findings for ITC pN0 (i+)(sn) No SLN metastasis histologically, positive morphological findings for ITC pN0 (mol−)(sn) No SLN metastasis histologically, negative nonmorphological findings for ITC pN0 (mol+)(sn) No SLN metastasis histologically, positive nonmorphological findings for ITC From Alkureishi LWT, Alvarez JA, Britten AJ, Gray HW, et al. Joint practice guidelines for radionuclide lymphoscintigraphy for sentinel node localization in oral/oropharyngeal squamous cell carcinoma. Eur J Nucl Med Mol Imaging. 2009;36(11):1915–1936. Reprinted with permission from Springer

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Melanoma The addition of immunohistochemical (IHC) techniques to standard H&E examination has been shown to increase melanoma detection rates by at least 10% [72], and a number of sectioning/staining protocols have been described in an effort to maximize detection rates while minimizing unnecessary workload. Some authors have advocated examination of only the central portion of the lymph node, based on the suggestion by Cochran et al. that the vast majority of micrometastases occur centrally [73], while other suggested protocols have included sectioning of the entire node into 1 mm slices [74], or examination of one half of the SLN using a combination of histology and immunohistochemistry, and the other half using RT-PCR with a variety of probes [75]. RT-PCR detection of occult metastatic deposits is an attractive technique, potentially reducing the cost and labor associated with SLN evaluation. However, disadvantages include its destructive nature, and positivity rates of up to 70% in some studies [76]. False positives may be due to capsular or trabecular nevus cells, nerves, or macrophages. In a recent report by Cook et al., utilizing an extended stepwise study of bivalved nodes with immunohistochemistry, the discrepancy between detection rates using histology/IHC and RT-PCR was found to be only 3–5%. Nevertheless, the exact role of RT-PCR remains to be fully elucidated and the authors therefore recommend the routine use of their extended histology/IHC protocol, which sections deeper into the periphery of the node, until further data become available [70]. This protocol is currently recommended by the EORTC, and is illustrated in Fig.  16.3. Briefly, the sequence involves bivalving the formalin-fixed SLN, embedding in paraffin, and sectioning at 50 mm intervals to a total depth of 250 mm. Several sections are taken at each interval, and are alternately stained with H&E, S100 and/or HMB45 for IHC. Sections found positive by IHC are compared with adjacent H&Estained sections in order to confirm the presence of viable tumor cells. “Spare” sections were stored for future use or stained with additional investigational antibodies, such as Pan Melanoma Plus (Biocarta). The use of this extended sectioning protocol results in thorough evaluation of the central 700–800 mm of each SLN, and is thought to represent the best balance between sensitivity, cost-effectiveness and pathologist workload [70]. For SCC, there remains considerable debate regarding the optimal method for sectioning SLNs. Current recommendations were formulated during the Second International Conference on Sentinel Node Biopsy in Mucosal Head and Neck Cancer in 2003, and are included in the recent joint guideline published by the European Association of Nuclear Medicine (EANM) and European Sentinel Node Trial (SENT) committee [54, 71].

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Pathologist Dissection

First full section

Section 1 – H&E 2 – S100 3 – spare

(+50µm)

50µm gap 4 5 6 7 8 9

(+100µm)

– – – – – –

H&E S100 HMB45 Pan Melanoma Plus spare spare

50µm gap 10 – H&E 11 – S100 12 – spare

(+150µm)

50µm gap 13 – H&E 14 – S100 15 – spare

(+200µm)

50µm gap 16 – H&E 17 – S100 18 – spare

(+250µm)

50µm gap 19 – H&E 20 – S100

Fig. 16.3  Extended stepwise examination of bivalved SLNs with immu­ nohistochemistry using S100 and HMB45 stains

SLNs less than 2 mm in longest dimension are processed whole while those measuring 2–5  mm should be bivalved and both halves processed en face. Nodes greater than 5 mm are cut into 2 mm slices, and each slice processed en face. A section from each slice is stained with H&E, and positive nodes/slices result in upstaging of the patient. Step-serial sectioning (SSS) at finer intervals of 150  mm (six sections per interval) should be carried out for SLNs found negative after initial sectioning, and these are H&E stained and examined as before. Finally, SLNs that remain negative are

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subjected to immunohistochemical (IHC) staining with pancytokeratin antibody (AE1/AE3 or MNF116). The combination of SSS and IHC has previously been shown to detect an additional 10% of occult/micrometastatic deposits compared with H&E alone [33]. If no disease is found following H&E and IHC staining, the lymph node is considered free of tumor. For SLNs with positive IHC staining, the positive section must be compared with the immediately adjacent serial section in order to avoid false-positives due to nonviable tumor cells, artifacts and/or inclusion of other cell types [54]. The use of intraoperative frozen section analysis of SLNs offers the potential advantage of avoiding a second anesthetic for SNB-positive patients, but has traditionally been avoided due to concerns regarding freezing artifacts and loss of tissue. More recently, these views have been challenged by a number of authors who report excellent results using the technique, with only 10–17% of SNB-positive patients requiring a second procedure [35, 77, 78]. However, the technique has not yet gained universal acceptance. Novel techniques, such as imprint cytology [79] and intraoperative real-time genetic evaluation, [80] currently remain under investigation.

Merkel Cell Carcinoma Pathologic evaluation of the sentinel nodes in MCC is similar to that of melanoma, though no standardized protocol has yet been adopted. The differences lie mainly in the type of stepserial sectioning, which varies from 2–3 mm slices [81] to 1  mm slices with multiple 200  mm sections per slice [82], and the use of anti-CK-20 staining (Dako Corp, Carpentaria, Calif.) in place of S100/HMB-45 for immunohistochemistry. CK-20 is well established as the most sensitive and specific marker currently available for the detection of MCC [83].

The Role of SNB in Current Practice Melanoma Following the initial reports of SNB for cutaneous ­melanoma using blue dye only, technical difficulties and the significant learning curve associated with the procedure led to variable technical success rates ranging from 60 to 80% [46]. Subsequently, the introduction of radio-labeled tracer ­injection, preoperative LSG and intraoperative gammaprobe guidance led to significant improvements in identification rates to greater than 90%, and the use of both blue dye and radiotracers quickly gained acceptance [36, 59, 84].

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Since then, the technique of SNB has been demonstrated to accurately predict the disease status of the remaining nodal basin in a number of landmark studies of cutaneous melanoma (all sites) [48, 85, 86]. The presence of metastases within SLNs has been demonstrated to be the most accurate predictor of outcome in melanoma patients without clinical lymph node involvement [87], and there is now some evidence to suggest that early lymphadenectomy following a positive SNB may confer a small but significant survival benefit over lymphadenectomy for nodal recurrence, albeit based on subgroup analysis (data from all sites) [88, 89]. As a result, SNB is now widely regarded as the gold standard for staging the lymphatic basins of intermediatethickness melanoma patients without clinical evidence of nodal involvement [46]. The primary indication for lymph node staging in this population is a primary tumor greater than 1  mm in Breslow thickness, though SNB should also be considered for thinner tumors in the presence of high-risk features, such as ulceration, high mitotic rate, or Clark level IV/V [46, 87]. In the head and neck, the prognostic significance of sentinel node status is less clear, with SLN-negative patients demonstrating a 5-year disease-free survival rate of only 55% in one report. In their review of the existing head and neck melanoma literature, the authors noted false-negative rates in excess of 10% in 12 of 21 studies, and suggested that this high false-negative rate may contribute to the poor survival they observed in their series [90]. Similar results were described in the large Sunbelt Melanoma Trial, where false-negative rates were 12% for the head and neck, compared with 2–3% for other sites [37]. However, this view has been challenged by Civantos et al., who contended that surgeons with a subspecialty focus on the head and neck may achieve negative predictive values comparable to the 98.2% for cutaneous malignancies and 92% for oral cancer described in their series of 106 patients with head and neck malignancy [91]. Concluding their review, Tanis et  al. stated that there is currently no conclusive survival advantage for either elective lymph node dissection or SNB in patients with intermediate thickness melanoma of the head and neck; however, the benefits of SNB may potentially justify its use in this patient population. These benefits include early prognostic information for patient and physician, reduced tumor load due to earlier lymphadenectomy, and the possibility of a survival advantage based on subgroup analysis [90]. A variety of micromorphometrical parameters of SN tumor deposits have been used in an attempt to determine the likelihood of further disease in the remaining nodal basin, such as tumor penetrative depth from the central plane, location within the node, and size. The potential applications for these measurements would include guidance of the decision to proceed with formal lymphadenectomy and prediction of survival.

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For example, the knowledge that only 10–30% of patients with positive SLNs are found to have additional positive “non-SLN” nodes following lymphadenectomy has led some authors to suggest that formal lymphadenectomy may not be required in patients with SLN deposits <0.1 mm in size [92]. However, the promising results reported in some series have not been universally reproduced in other studies, and as a result the prognostic significance of tumor burden in the sentinel nodes has not yet been fully elucidated. In the meantime, it is recommended that all patients with detectable disease in the sentinel nodes be treated as SN-positive and offered formal lymphadenectomy [46, 75].

Future Application of SNB for Melanoma of the Head and Neck For melanoma, SNB is well established as a staging tool for patients with intermediate thickness primary tumors, and for selected patients in other groups. The main questions now focus on the optimal management of SNB-positive patients, and this is currently unclear. The MSLT-2 trial is a prospective randomized controlled trial, comparing the outcomes of completion lymphadenectomy and observation alone for SNB-positive patients [48] while further upcoming studies aim to randomize SNB-positive patients to receive completion lymphadenectomy or therapeutic irradiation [93], and interferon-alpha alone or interferon-alpha and completion lymphadenectomy [94]. Until the results of these studies become available, the recommended management of all SLN-positive patients is completion lymphadenectomy. In addition, the differences in technical success and false negative rates for SNB in the head and neck compared with other sites suggests that the results of large-scale prospective RCTs reporting all-sites melanoma data may not be immediately applicable to the head and neck population. Therefore, similar prospective trials tailored specifically to this patient group are required before definitive conclusions regarding optimal management can be reached.

Oral/Oropharyngeal Squamous Cell Carcinoma Validation of the SNB technique for patients with early oral/ oropharyngeal SCC has, until recently, involved staging patients with SNB, followed by immediate elective neck dissection [34, 49, 95]. From these studies, it has been demonstrated that SNB may be safely and successfully applied to patients with T1 or T2 disease and cN0 necks [33, 54]. The vast majority of the tumors studied to date are located in

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the oral cavity or accessible oropharynx and, while some reports do exist of SNB for other locations, such as the hypopharynx and supraglottic larynx [96], the status of the technique should remain “investigational” in these sites until further data becomes available. Furthermore, the use of SNB may be limited in patients with larger tumors which may be difficult to completely surround with tracer injections and which may ultimately require a neck dissection for tumor access or reconstruction purposes [51]. The promising results of these validation studies, demonstrating false negative rates of approximately 5%, have led some centers to over SNB as the sole staging tool for selected patients with early OSCC, with only those patients found SNB-positive going on to receive completion lymphadenectomy [33, 35]. The applications for SNB in early OSCC include staging of the ipsilateral cN0 neck, staging bilateral cN0 necks for tumors with ambiguous drainage (i.e., midline), and staging the contralateral cN0 neck for a midline tumor with an ipsilateral cN+ neck. Other applications, including the use of SNB for patients with recurrent primary tumors or following prior treatment to the neck, remain under investigation. At the time of writing, there have been two large prospective clinical trials reported, examining SNB in this patient population [33, 35]. The interim results of a European multicenter trial involving patients from six centers were published in 2004, and demonstrated a 93% SN identification rate and 93% sensitivity in 134 patients undergoing SNB-assisted-END or SNB-alone for cT1/2 cN0 OSCC [33]. The 5-year follow-up for this population revealed one further nodal recurrence, giving an overall sensitivity of 91% at 5 years [97]. The identification rate and sensitivity were found to be significantly lower for patients with floor-of-mouth tumors, which the authors attribute to the technically challenging access to these tumors and close proximity to the first echelon lymph nodes. The authors concluded that SNB can safely be used as the sole staging tool for the majority of patients with early OSCC, but advise caution when evaluating floor-of-mouth tumors [33, 97]. Similar outcomes were reported by Stoeckli et al. in the largest single-institution series reported to date [35]. The authors reported a 98% identification rate and 94% negative predictive value in a series of 51 patients undergoing SNB alone for cT1/2, cN0 OSCC. The SENT is a large prospective study, incorporating the data from these two previous studies and several additional European centers. An interim analysis of this dataset, focusing on SLN-positive patients, was reported at 27 months of follow-up [98]. Of 72 patients (86 neck sides) undergoing completion lymphadenectomy for a positive SNB, 42% were found to harbor additional disease in the neck dissection specimen. Fifty-two percent of these additional positive nodes were located in the same neck level as the positive SLN, and only

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4% were located out with the two adjacent neck levels. The authors conclude that it may be reasonable to limit therapeutic lymphadenectomies following positive SNB to three levels – one above and one below the positive SLN – potentially further reducing the morbidity associated with treatment of the neck.

Cutaneous SCC of the Head and Neck For patients with cutaneous SCC, the rate of nodal metastasis is much lower, ranging from 0.3 to 16% [99, 100]. As a result, SNB has not been well studied in this patient group. As part of a larger series of multiple tumor types, Civantos et al. undertook SNB in a series of ten patients with “highrisk” cutaneous SCC, and detected occult nodal disease in only one patient. The authors concluded that further study is required to determine the most appropriate management strategy for these patients [91].

The Future of SNB in Oral/Oropharyngeal SCC SNB provides the means for accurate and minimally invasive pathologic staging of the cN0 neck in patients with early OSCC. However, the exact role of SNB in the management of this patient group has yet to be fully elucidated and as a result, the technique has not yet gained universal acceptance. It is hoped that the upcoming results of the SENT trial and American College of Surgeons’ Oncology Group (ACOSOG) Z0360 validation study [101] will provide the foundations for randomized phase III studies comparing SNB-alone with elective neck dissection, which currently remains the gold standard in most centers [101].

Merkel Cell Carcinoma MCC is a rare, highly aggressive neuroendocrine tumor arising from the Merkel mechanoreceptor of the skin. It is associated with an overall 5-year survival of 30–64%, with a high incidence of local recurrence, regional lymph node involvement, and distant metastasis [102, 103]. In part due to the rarity of this tumor, there is no consensus on the current standard of care for management. Excision of the primary tumor may require wide margins for elective local control [104], or the addition of adjuvant radiotherapy if smaller margins are used [105]. In some series, radiotherapy alone has been shown to achieve similar local control rates to primary excision [106]. Elective treatment of the lymph

L. Alkureishi and G.L. Ross

nodes should be strongly considered due to reported nodal recurrence rates of up to 76% of stage I MCC patients in some series [107]. Prophylactic lymph node dissection appears to improve regional control, but does not lead to improved survival [108]. As a result, there is some disagreement regarding the utility of prophylactic node dissection in this population [82, 109]. Similarly, the utility of SNB in patients with early stage MCC is a topic of considerable debate. Advocates of the technique contend that SNB can help identify patients with occult nodal disease, demonstrate aberrant drainage patterns, and may prevent unnecessary neck dissection, parotidectomy and/or irradiation [81, 82]. In an exhaustive review of the existing literature, Mehrany et  al. considered 60 patients undergoing SNB for MCC, and reported that SNB-positive patients were 18.9 times more likely to have nodal recurrence compared with SNB-negative patients after a median follow-up of 7 months [110]. Schmalbach et al. subsequently described a series of ten patients, eight of whom were found SNB-negative. After median follow-up of 34 months, nodal recurrence was observed in only one patient (12%), leading the authors to conclude that SNB is a safe and reliable technique for staging MCC [81]. However, these findings are at odd with a subsequent report by Warner et al., who found that SLN status is not an accurate predictor of locoregional recurrence in a series of 17 patients with MCC and a median follow-up of 16 months. The authors instead advocate the use of local and regional radiotherapy as a means of obtaining elective infield disease control [111]. Similarly, in a series of 23 patients undergoing SNB after previous excision of MCC, a nodal recurrence rate of 33% was noted in the SNB-negative group, leading the authors to question the prognostic value of SNB for MCC [112]. A smaller series of ten patients was recently reported by Schnayder et  al., with six patients found SNB-negative. Of these, one patient developed nodal recurrence during the follow-up period (median 24 months). The authors concluded that, in this patient population with very high rates of occult micrometastatic lymph node involvement, the true utility of SNB may be ensuring that all at-risk nodes are adequately addressed, even in cases of “aberrant” drainage, e.g., to intraparotid lymph nodes or the contralateral lymphatic basin. Furthermore, SNB may allow for accurate staging in patients who are reluctant to undergo formal lymphadenectomy [82]. As with melanoma and SCC, the true prognostic significance of submicroscopic lymph node metastases, which are reported to occur in up to 100% of MCC patients, remains unclear [113]. Further study will be required to clarify the exact role of SNB in this population. In the USA, the National Comprehensive Cancer Network (NCCN) currently recommends SNB for all patients presenting with previously untreated, localized stage I disease (NCCN Clinical Practice Guidelines in Oncology, v.1.2004).

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Complications of Sentinel Node Biopsy The steep learning curve, technical difficulty and minimally invasive approach of SNB may potentially lead to a higher risk of complications compared with formal lymphadenectomy; principally, damage to the facial or spinal accessory nerve. In addition, the requirement for a completion lymphadenectomy in SLN-positive patients represents a second procedure in an inflamed, recently operated surgical field, theoretically contributing to the risk of iatrogenic injury [91]. However, in experienced hands the incidence of complications following SNB is reported to be as low as 1% [37, 114]. For SLNs located in the region of the parotid gland, some authors advocate careful dissection and enucleation of the sentinel nodes. However, high rates of facial nerve paresis in selected studies have led some authors to recommend superficial parotidectomy over biopsy alone [37, 115].

Summary SNB represents a useful tool for staging the cN0 lymphatic basins in patients with selected head and neck malignancies. For patients with melanoma, SNB is widely accepted as the gold standard staging tool for patients with intermediate thickness tumors, and may also be useful for patients in other groups. However, questions remain with regards to the optimal management of SNB-positive patients and the prognostic significance of very small tumor deposits. For the management of patients with early OSCC, SNB has not yet gained universal acceptance as a sole staging tool, and the results of ongoing large prospective trials are awaited in order to better understand its true role. Finally, the prognostic value of SNB for MCC has been questioned, and its utility may ultimately be limited to improvements in staging. Sentinel node biopsy has improved staging and has led to a more appropriate selection of oncological therapies. It is essential that sentinel node biopsy be performed in oncological centers by validated teams of surgeons, pathologists and nuclear medicine physician’s with rapid access to oncologists and clinical trials on site.

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Chapter 17

Approaches to Supportive Care Barbara A. Murphy

Abstract  Head and neck cancer is associated with ­substantial symptom and function loss. It is critical to understand the depth and breadth of issues face by patients in order to maximize quality of life. Symptoms and functional deficits may be secondary to either the cancer or its treatment. The mechanism of toxicity varies depending on the extent of tumor involvement, the site of tumor, treatment modality, and host factors. Toxicity is usually categorized as acute (occurring within 3 months of therapy) or late (occurring 3 months or after therapy). In addition, it is also important to distinguish local versus systemic toxicities. Although head and neck cancer therapy is associated with significant system effects, data pertaining to these toxicities are lacking. Thus, this chapter reviews the selected critical supportive care issues localized to the head and neck region. This includes: mucositis, nutrition, dysphagia, xerostomia and hyposalivation, oral health issues, and radiation dermatitis. Keywords  Head and neck cancer • Symptoms • Function • Pain • Mucositis • Nutrition • Dysphagia • Xerostomia • Trismus • Dental • Sialorrhea • Dermatitis

Introduction Head and neck cancer and its treatment are associated with clinically significant symptom burden, alterations in function, and decrease in quality of life [1]. Due to the frequent compromise of structures which are critical for functions such as speech, swallowing, and breathing, supportive care has always been a critical albeit underappreciated component of head and neck cancer therapy. More recently, the role of supportive care has been highlighted due to a number of

issues, including: the increased use of aggressive combined modality therapies which are associated with an increase in acute and late effects, the increasing numbers of survivors who are living with the late effects of therapy, and the recognition that without appropriate management, the cost of acute and late effects to both the patient and society can be staggering.

Mechanism of Toxicity Acute Tissue Damage Normal tissues may be damaged by either cancer or its treatment. Surgical damage results from removal of the cancer and a surrounding rim of normal tissue. The degree to which surgical resection causes morbidity is related to the amount of tissue removed, the site of the tissue removed, and the ability to use reconstructive techniques to ameliorate the effect of normal tissue loss. Vascular and neurologic damage may contribute to surgical morbidity. Radiation therapy results in DNA and non-DNA damage to tissues within the radiation therapy port. The tissue damage initiates a sequence of biologic pathways that are involved in wound healing and tissue repair. In addition, both surgery and radiation may be associated with systemic effects [2] such as fatigue, deconditioning, pain, and altered mental status. These systemic effects of therapy are in part the result of: (1) proinflammatory cytokines and chemokines released as a component of acute tissue inflammation, (2) the humoral and neurologic stress response, and (3) drug-related toxicities.

Late Tissue Damage B.A. Murphy (*) Pain and Symptom Management Program, Internal Medicine, Vanderbilt Ingram Cancer Center, 777 Preston Research Blvd, Nashville, TN 37215, USA e-mail: [email protected]

Tissues damage by surgery or radiation must undergo repair. During the repair process, damaged tissue may be replaced by normal functioning tissue. Alternatively, tissue repair

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mechanisms may cause replacement of normal tissue with fibrotic tissue. Fibrosis results from a chronic inflammatory process that involves growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines [3]. It is manifested by excessive deposits of extracellular matrix by fibroblasts with resultant abnormal tissue architecture. There are three histopathologic phases of fibrosis: (1) chronic inflammation without fibrosis, (2) active fibrosis with dense myofibroblasts, and (3) late fibrosis with associated atrophy and decrease in parenchymal cells [4]. Tissues become noncompliant, contracted, and atrophic resulting in altered function and significant symptom burden. Thus, fibrosis plays a critical role in the development and manifestations of late tissue damage in the head and neck cancer population.

Specific Acute and Late Effects of Therapy Mucositis Mucositis is a process that results from chemotherapy and radiation-induced damage to the mucosa and underlying soft tissue. Recent studies have helped to elucidate the complex biologic mechanism underlying the tissue repair response and its associated manifestations [5, 6]. The clinical hallmarks of mucositis are erythema and ulceration of the mucous membranes. In addition, the underlying soft tissue may become swollen and edematous. There are a number of systems that have been used to grade mucositis (Table 17.1), the most frequently used systems are the Common Toxicity Criteria and the World Health Organization (WHO) Toxicity Criteria. The Common Terminology for Adverse Events 3.0

Table 17.1  Mucositis scoring systems 1

contains two separate criteria for grading mucositis: (1) direct visualization of lesions and (2) assessment of the functional impact of mucositis. The WHO criteria combine symptoms, function, and mucosal pathology into one single measure. Thus, it should be noted that there may be differences in how patients’ mucositis is graded based on the toxicity criteria used. Unfortunately, underreporting of the frequency and severity of mucositis by health care providers is common. To avoid some of the pitfalls associated with health care provider for mucositis assessment, a number of tools have been developed which use patient-reported outcomes to measure mucositis severity and symptom burden. The most commonly used tool is the Oral Mucositis Questionnaire Head and Neck (Daily and Weekly versions). Originally developed for use in the transplant setting, the OMQ-HN has been demonstrated to be a valid and reliable tool for assessment of mucositis-related symptom burden [7]. The questionnaire focuses on mucositis-related pain and function loss. The risk for the development of mucositis is highly variable and is based on a number of predictive factors. It has long been known that primary site, radiation dose, radiation schedule, and port size correlate with the extent and severity of mucositis [8, 9]. Although radiation parameters are clearly important, the most powerful predictor for the development of severe mucositis is the use of concurrent chemotherapy. In a retrospective review of 33 clinical treatment trials, Trotti reported that the incidence of grade 3 and 4 mucositis rose from 25 to 40% with radiation therapy alone to 60–100% in patients treated with chemoradiation [10]. In addition to an increase in the incidence of oral mucositis, the use of concurrent chemoradiation has been noted to increase the duration of mucositis [9]. Although tumor and treatment-related factors clearly predict mucositis outcomes, the role of patient

2

3

4

5

WHO [110]

Erythema and soreness; no ulcers

Ulcers; able to eat a solid diet

Ulcers; requires a liquid diet

NA

CTCAE v 3.0 [111] (clinical exam)

Erythema

Patchy ulcerations or pseudomembranes

Confluent ulcerations or pseudomembranes

Ulcers; not able to tolerate a solid or liquid diet; requires IV of tube feeding Tissue necrosis; significant spontaneous bleeding, lifethreatening Symptoms associated with life-­threatening consequences

Symptomatic and Symptomatic but can eat Minimal symptoms; unable to adequately and swallow modified normal diet; minimal aliment or hydrate diet; respiratory respiratory symporally; respiratory symptoms interfering toms but not symptoms interferwith function but not interfering with ing with ADL with ADL function WHO World Health Organization, CTCAE 3.0 Common Terminology Criteria for Adverse Events

CTCAE v 3.0 (functionalsymptomatic)

Death

Death

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characteristics remains unclear. Numerous patient-related factors have been studied, yet epidemiologic data is lacking to clearly link mucositis outcomes with specific demographic factors [11]. The contribution of genetics factors is unknown at this time. Mucositis-related symptoms usually begin to manifest themselves within 2–3 weeks after radiation therapy is initiated. Initial complains include throat irritation and pain on swallowing. By week 5 of radiation, mucosal lesions have worsened substantially leading to moderate-to-severe pain. Unfortunately, mucositis-related pain is often refractory to opioid analgesics. Physical exam findings and symptoms usually peak within 2–3 weeks of completing therapy and gradually subsides thereafter. It is not uncommon for symptoms and ulcerative lesions to persist for 2–3 months after radiation therapy is completed. Occasionally, patients with developed ulcerative lesions fail to heal or heal over a protracted period of time. In this population, hyperbaric oxygen therapy or treatment with pentoxifylline may be attempted, however, data confirming efficacy is lacking. Mucositis results in a number of adverse outcomes [12]. First and foremost, severe mucositis results in treatment breaks that may compromise disease control and survival [9, 10]. Second, mucositis is associated with significant symptom burden and alterations in function. The most common mucositis-related symptom is pain. Pain results in decreased speaking, swallowing, eating, and oral care [7]. Pain is worse on swallowing, thus leading to decreased oral intake [13]. For many patients, the pain becomes severe enough that adequate oral alimentation is not possible and a feeding tube is required. Data from the Longitudinal Oncology Registry for Head and Neck Cancer indicated that feeding tubes were placed in 59% of patients at academic centers and 48% of patients at community centers within the USA (p = 0.001) [14]. Finally, mucositis results in an increase in the use of health care resources and associated increased cost of care [15]. The cost differential between patients with and without radiation-induced mucositis is variable based on the patient population and the severity of the mucositis. For patients with severe mucositis, the cost increment has been reported between $6,000 [9] and $18,000 [16]. Due to the high cost, investigators have attempted to identify effective preventive and treatment strategies for radiation-induced oral mucositis. A wide array of treatment interventions has been tested. To date, none have clearly demonstrated a marked impact on the incidence or duration of grade 3 and 4 mucositis. The Multinational Associate for Supportive Care in Cancer has a standing committee that has developed and updated an evidence-based guideline for the treatment, prevention, and palliation of mucositis. Their recommendations include the following: oral care

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p­ rotocols, adequate use of pain medications, and the use of conformation radiation techniques to minimize mucosal injury. Updated recommendations can be found on their Web site at www.mascc.org.

Swallowing Abnormalities Dysphagia is one of the most common and concerning sequelae of head and neck cancer and its therapy. The normal swallowing mechanism is complex, requiring coordination of over 25 pairs of muscles [17], as well as an intact nervous system that mediates both voluntary and involuntary swallowing maneuvers [18]. The four phase of swallowing are: (1) the oral preparatory phase (food bolus formation), (2) the oral phase (bolus transported to the pharynx), (3) the pharyngeal phase (reflex closure of the larynx to prevent aspiration, coordinated contraction of the pharyngeal constrictors, and relaxation of the cricopharyngeus muscles) [19, 20], and (4) the esophageal phase (peristalsis of bolus into the stomach). Abnormalities in any of the above functions may result in clinically meaningful dysphagia. Symptoms that indicate the presence of dysphagia are listed in Table 17.2. When dysphagia is suspected, a formal functional assessment is indicated. The clinical evaluation of swallowing (CES), which should be performed by an experienced Speech-Language Pathologists (SLP) [21], includes the following components: (1) identification of swallowing abnormalities, (2) recommendations for additional testing, (3) development of a treatment plan when indicated, (4) consultation with dieticians to develop a nutritional plan that is safe, and (5) assessment of aspiration risk. The SLP may ­recommend instrumental studies to assess swallowing function. The modified barium swallow study (MBSS) is a videofluoroscopic exam of the oral and pharyngeal function that identifies swallowing impairments and aspiration [22]. Food boluses of differing sizes and consistencies are assessed Table 17.2  Triggers for dysphagia evaluation [112, 113] • Inability to control food, liquids, or saliva in the oral cavity • Pocketing of food in cheek • Excessive chewing • Drooling • Coughing, choking, or throat clearing before, during, or after swallowing • Abnormal vocal quality after swallowing – “wet” or “gurgly” voice • Build-up or congestion after a meal • Complaint of difficulty swallowing • Complaint of food “sticking” in throat • Nasal regurgitation • Weight loss

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leading to appropriate dietary recommendations as well as testing of compensatory measures that may enhance swallow efficacy and safety. Standard compensatory measures include postural techniques, increased sensory input, and voluntary swallowing maneuvers. In addition, direct visualization of the structures and functioning of the pharynx and larynx can be done using the Flexible Endoscopic Evaluation (FEES) [23] allowing identification of issues such as: (1) premature spillage, (2) pooling, (3) laryngeal penetration, (4) aspiration, and (5) laryngopharyngeal reflux [24]. Dysphagia related to physiologic damage by an infiltrative cancer may be present at the time of diagnosis; however, it is more commonly due to the acute and late effects of surgery and radiation therapy. Resection of structures that are critical for normal swallowing function or surgically induced neurologic damage may result in postoperative dysphagia. Studies have demonstrated that the extent of dysfunction correlates with the site and extent of tissue resected [25, 26]. Acutely, postoperative dysphagia may be exacerbated by tissue edema and pain, while long-term tissue fibrosis and scar may contribute to persistent or deteriorating swallow function over time. Acute dysphagia secondary to radiation therapy induced tissue damage manifested by painful mucositis, soft tissue edema, and thick mucous production. As the soft tissues and mucosa heal, scaring may take place resulting in the formation of fibrotic, noncompliant tissues [3]. Fibrosis may result in altered function including abnormal swallowing. Eisbruch identified “dysphagia/aspiration-related structures” (DARS) [27]. When these structures sustain acute and chronic damaged secondary to radiation, patients are at high risk for dysphasia and aspiration. Minimizing radiation to these structures using radiation techniques such as intensity modulated radiation therapy (IMRT) has been shown to improve swallowing outcomes [28, 29]. It should be noted that the use of concurrent chemotherapy with radiation therapy is associated with an increase of acute mucosal and soft tissue damage [30]. Although the relationship remains difficult to prove [31], increased acute toxicities are postulated to result in increased late effects; thus, explaining the clinical observation that patients receiving aggressive CCR regimens have a higher incidence of late effect dysphagia and long-term feeding tube dependence. Stricture formation is an extreme fibrotic process which is generally noted in the upper esophagus. It may contribute to or be wholly responsible for a patient’s dysphagia. The majority of patients with upper esophageal stricture formation received high doses (>60 Gy) of radiation to the involved structures [32]. The use of concurrent chemoradiation does appear to increase the risk for strictures [33]. Usually identified on MBSSs, strictures may be treated with endoscopic balloon dilatation. Data would indicate that this technique is

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successful in a high percentage of patients; however, repeat dilation is often required [34]. There are numerous sequelae of dysphagia, of which aspiration is the most concerning. Acutely, aspiration may result in pneumonia. In the head and neck population, particularly those receiving particularly myelosuppressive chemotherapy, pneumonia has been associated with significant morbidity and mortality [35]. Long-term chronic aspiration can result in pulmonary fibrosis and permanent lung damage [36]. Moderate dysphagia may result in altered dietary intake. In some patients, this may lead to poor diet quality and dietary inadequacies [37, 38]. Patients with severe dysphagia and/or aspiration may require a permanent feeding tube in order to ensure adequate and safe nutritional intake [31]. Predictive factors for long-term feeding tube dependence includes: oro/hypopharyngeal primaries, stage III/IV disease, flap reconstruction, current tracheotomy, chemotherapy or increased age [39].

Nutrition Nutrient intake is often compromised in head and neck cancer patients’ either due to symptoms from their cancer or its treatment. Factors that may contribute to malnutrition include: (1) alimentary track obstruction or dysfunction, (2) radiation-induced acute effects such as mucositis, mucous production, and tissue edema, (3) chemotherapy side effects such as anorexia, nausea, and vomiting, (4) a history of substance abuse with associated nutrient deficiencies, (5) socioeconomic factors that inhibit patients from obtaining nutritionally replete diet or supplements, and (6) cancer cachexia syndrome with associated metabolic abnormalities that favor proteolysis. Overall, malnutrition is seen in 30 and 50% [40, 41] of head and neck patients; however, the numbers are substantially higher in patients with locally advanced disease [42, 43]. Weight loss is associated with numerous adverse outcome measures including: surgical complications [40], immune function [40], survival [41, 44, 45], and quality of life [45]. Thus, ongoing nutritional assessment is critical in all patients with head and neck cancer. At diagnosis, a baseline nutritional assessment is vital for all head and neck cancer patients [46, 47]. This should include an accurate weight, weight loss history, and identification of barriers to adequate nutritional intake. Patients with a stable weight and adequate oral intake may be monitored prospectively. Patients with critical weight loss (see Table 17.3) should be seen by a dietician in order to generate an appropriate nutritional plan. Basal energy expenditure (BEE) can be calculated using the Harris Benedict equation [48] which takes weight, height, and age into consideration.

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Table 17.3  Definition of critical weight loss [114] Cumulative weight loss and time course Significant weight Severe weight Time course loss (%) loss (%) 1 week 1 month 3 months 6 months

£2 £5 £7.5 £10

>2 >5 >7.5 >10

Of note, the physiologic stress of therapy may substantially increase a patient’s caloric requirement. This should be taken into account when counseling patient regarding caloric and protein goals. Men: BEE = 66.5 + (13.75 kg) + (5.003 cm) - (6.775 ´ age), Women: BEE = 655.1 + (9.563 kg) + (1.850 cm) - (4.676 ´ age). The placement of a feeding tube may be necessary in order to ensure adequate nutritional intake. In the postoperative population, a nasogastric tube may be used in patients who are expected to have dysphagia of limited duration. For those patients who are expected to have protracted or permanent dysphagia, a percutaneous endoscopic gastrostomy (PEG) tube is usually placed [49, 50]. The role of feeding tubes in patients undergoing radiation therapy remains controversial. It is clear that radiation therapy results in painful mucositis, edema, and mucous, which decreases intake and contributes to treatmentassociated weight loss. Reports indicate that the weight loss associated with radiation therapy is as high as 10% [51]. Data clearly demonstrate that the use of prophylactic feedings tubes reduces weight loss during and immediately after radiation therapy has been completed [52, 53]. Furthermore, the complication rate is low and most complications are generally minor [54]. However, there is concern that feeding tubes result in disuse atrophy and late effect dysphagia [31, 50]. Regardless of when a feeding tube is placed, posttube placement patients should be encouraged to continue to swallow as tolerated, to comply with swallowing exercises, and to wean off the feeding tube as quickly posttreatment as is feasible. Once placed, the health care team work with the patient and caregiver to ensure that an appropriate nutritional plan is established and followed. It is important to recognize that the placement of a feeding tube does not in and of itself guarantee adequate caloric intake. The proper use and maintenance of a PEG or NG tube is complex and requires proper education and training. The patient’s ability to master the use of feeding tube may be diminished by mental status changes, generalized weakness, and debility. Caregivers frequently spend considerable time helping in the care of head and neck cancer patients with feeding tubes [55]. Feeding tubes are associated with a number of management challenges. One of the most common issues difficult to

achieve is the intake of the desired amount of formula. This is often secondary to gastrointestinal dysmotility. Dysmotility may result from medications (such as opioids), electrolyte imbalance, decrease in activity level, dehydration, and the physiologic stress response. Symptoms of dysmotility include: nausea and vomiting, early satiety, and bloating. Prokinetic agents such as metaclopromide can increase gastric motility and ameliorate symptoms. Tubes must be inspected routinely to evaluate for infection, dermal irritation, leakage around the tube, and damage to the tube which requires repair or replacement. Upon completing therapy, patients should be encouraged to transition to oral nutrition as quickly as possible. That being said, many patients experience late effect dysphagia. For some patients, dysphagia is of sufficient severity that oral alimentation is not feasible; thus leading to long term or permanent feeding tube dependence. For others, dysphagia may be less severe, resulting in altered food choices. The dietary adaptations that patients make in order to maintain an oral diet may be adaptive or maladaptive depending on the resulting nutrient intake. When dietary adaptations result in dietary inadequacies, supplementation is indicated. It should be noted that dysphagia associated with nutritional deficiencies may persist long term; thus, ongoing and periodic assessment by a dietician should be included in routine follow-up for head and neck cancer patients [37, 56]. Cachexia refers to a hypermetabolic state that is associated with proinflammatory cytokines [57]. It is associated with a number of symptoms including: anemia, weight loss, weakness, muscle, and fat wasting [58, 59]. Anorexia, which results from the loss of balance in the peripheral and central orexigenic and anorexigenic hormonal and neuropeptide signals, commonly occurs in patients undergoing active treatment and those with advanced disease [60]. To date, there is no convincing evidence for efficacy of any pharmacologic intervention for the treatment of cachexia. The French National Federation of Cancer Centers [61] has recommended the use of megestrol acetate, corticosteroids, and medroxyprogesterone for the treatment of anorexia. Data on the use of these agents in head and neck cancer are limited. In one randomized trial in patients treated with chemoradiation, the use of megace is increased appetite (p = 0.0001) and resulted in decrease of weight loss [62].

Xerostomia and Hyposalivation Xerostomia is the patient-reported symptom of dry mouth; hyposalivation is defined as a decrease in stimulated and/or unstimulated salivary flow. The normal unstimulated salivary flow is 0.3–0.5 ml/min and the normal stimulated flow rates

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are 1–2  ml/min. CTCAE 3.0 criteria for xerostomia and hyposalivation are as follows: grade 1 – symptomatic (dry or thick saliva) without significant dietary alterations or unstimulated flow rate of >0.2 ml/min; grade 2 – symptomatic and significant oral intake alterations (e.g., copious water, other lubricants, diet limited to purees and/or soft, and moist foods) or unstimulated flow rate of 0.1–0.2 ml/min; Grade 3 symptoms lead to inability to adequately aliment orally, IV fluids, tube feedings, or TPN indicated or unstimulated flow rate of <0.1  ml/min. While xerostomia is associated with discomfort and decreased quality of life [63–65], hyposalivation has been associated with a number of adverse oral health outcomes. Of note, the correlation between the subjective symptom of xerostomia and the objective measure of hyposalivation may be poor; thus, it is important to assess both outcome parameters. Saliva is a complex fluid of electrolytes, secretory ­proteins, and organic molecules [66]. It serves numerous physiologic functions that are integral to oral health including the following: lubrication of the mucous membranes, maintenance of the mucous membranes, aids in soft tissue repair, direct antibacterial effects, antiviral and antifungal effects, maintenance of pH, and maintenance of dental integrity [67]. Hyposalivation may result in increased symptom burden and functional loss (voice, swallowing, and sleep disturbance) as well as diminished oral health [68–72]. Although xerostomia and hyposalivation may be caused by a number of etiologic factors in the head and neck cancer population, the leading cause is radiation therapy induced damage to the salivary glands. Most patients note the development of symptoms within 2–3 weeks of initiating therapy [73]. Once therapy is completed, salivary gland function may return slowly over time. The severity of symptoms is related to the volume of salivary gland radiated [74]. Studies have shown that salivary gland damage is at least partially reversible when the total dose is 2,500– 3,000 cGy. Above that dose, xerostomia may be permanent. Thus, considerable research has been conducted to identify methods to prevent or limit radiation-induced xerostomia and hyposalivation. Approaches for the prevention of salivary gland damage from radiation therapy include: (1) surgical transplantation of the salivary glands out of the radiation port, (2) radiation techniques to minimize radiation-induced damage, and (3) pharmacologic techniques to prevent tissue damage. Salivary gland transfer is an technique during which the parotid gland is surgically transplanted to the submental space where it is shielded from radiation. Although numerous studies have demonstrated that this is a feasible and effective technique [75], it has not been broadly adopted. This may be due to the rapid increase in the use of IMRT as an alternative tissue sparing approach. Several pharmacologic agents have been investigated to determine their

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c­ apacity as cytoprotective agents in patient receiving ­radiation therapy to the salivary glands. The most extensively studied agent is amifostine, a free radical scavenger. In a meta-­analysis conducted by Sasse, amifostine was shown to modestly decreased acute and late effect xerostomia [76]. Furthermore, several small studies demonstrated that the use of amifostine resulted in improved dental outcomes [77, 78]. Use of IV amifostine was limited due to toxicity including nausea, vomiting, and hypotension. This led to the evaluation of ­subcutaneous administration which proved to be equally effective to IV administration but substantially less toxic [79]. Pilocarpine has also been evaluated as a potential cytoprotective agent to prevent radiation-induced xerostomia [80]. RTOG 97-09 randomized 245 patients with a planned radiation dose of ³50 Gy to the oral cavity/pharynx to either pilocarpine or placebo. Patients receiving pilocarpine had a significant increase in unstimulated salivary flow post treatment and at week 13. No improvement was noted in stimulated salivary flow rates or QOL measures [81]. No oral health outcomes were reported. Finally, intensity modulated radiotherapy (IMRT) is a technique that allows radiation to be directed at the tumor while minimizing the dose to normal tissue. While randomized control trials are limited [82], cumulative evidence supports the hypothesis that IMRT allows sparing of salivary tissue and decrease in late effect xerostomia without compromising the radiation dose to the tumor [83–85]. Once xerostomia develops, the clinician must direct attention to (1) assessment and minimization of long-term oral health implications of hyposalivation (see section on “Oral Health Issues” below), (2) maximizing residual salivary flow, and (3) maximizing patient comfort. Gustatory and pharmacologic stimulants may increase salivary flow. Commonly patients will use sugar-free lozenges or gum with some relief of symptoms. Pharmacologic agents include pilocarpine and cevamaline. Pilocarpine is a parasympathamimetic agent that functions as a nonselective muscarinic agonist. In a randomized trial of 207 patients with radiation-induced xerostomia, pilocarpine was associated with an increase in salivary flow, improved comfort, and improved speech [86]. A second agent, cevemaline, acts as a selective M3 muscarinic receptor agonist. Two large randomized trials demonstrated that cevemaline results in increased salivary flow rates; however, the effect on patient-reported symptoms was mixed [87]. A number of topical agents have been developed that are generally classified as “salivary substitutes” [88]. The efficacy of these agents is variable and patient specific. Patients should be encouraged to try several agents in appropriate dose and schedules to determine whether they receive benefit. For those patients who do not receive benefit from salivary substitutes, carrying a water bottle for frequent oral rinsing can provide temporary relief. The use of a humidifier, particularly at night, may diminish discomfort [89].

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Oral Health Issues Dental The major constituent of the dental enamel is calcium ­phosphate. Like bone, the enamel is constantly remodeling. Ideally, demineralization of the enamel surface is balanced by re-mineralization. However, if the balance sways toward demineralization, dental carries may develop. A number of factors predispose to demineralization, including an acidic milieu, lack of enamel substrates (calcium and phosphate), and cariogenic bacteria (streptococcus and lactobacillus species). Protective factors include fluoride treatment, ­calcium–phosphate paste/rinse, certain foods, and routine dental care. Radiation induces hyposalivation which in turn results in loss of salivary buffering capacity and a decrease in enamel substrates for re-mineralization. This predisposes to the development of carries. Postradiation dental carries can develop shortly after the completion of radiation and may progress very rapidly. Manifestations include demineralization, fracture of the enamel with chipping, and auto-­amputation of the tooth at the root. Even with aggressive dental intervention, it may not be possible to salvage dentition that ­manifests severe and rapidly progressing radiation carries. Thus, it is clear that oral health must be addressed aggressively throughout the trajectory of a patient’s treatment course and long term for survivors. Prior to the initiation of radiation therapy patients should undergo a thorough dental evaluation [90]. Nonviable teeth should be extracted 10–14 days prior to radiator to allow adequate healing. Patients must be educated extensively about oral health measures and compliance monitored on a routine basis. Patients should be instructed to brush after every meal. Oral rinses, such as baking soda gargles, may be used to buffer an acid pH. Patients should avoid acidic or sugar containing candy, drinks, or medications. Fluoride treatment should be utilized to enhance re-mineralization [91]. Chlorhexidine rinse may be used to minimize colonization with cariogenic bacteria. In small studies, posttreatment stimulation of residual salivary function with sialogogues has decreased late dental carries. Sialorrhea Patients commonly complain of “excess” mucous production. In a cohort of patients, salivary production may be normal but physiologic abnormalities such as dysphagia and obstruction prevent normal handling of secretions. In this group, treatment should be directed at maximizing control over secretions. On the other hand, patients may actually have an increase in salivary production or altered salivary

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texture leading to difficulty in managing secretions. Commonly, patients undergoing radiation therapy will complain of copious, thick sputum that is difficult to expectorate or swallow. Clearing secretions is hampered by painful mucositis, dysphagia, and pharyngeal edema. Treatment is directed at suppression of mucous production with pharmacologic agents such as scopolamine or atropine, thinning of mucous by the use of mucolytics, night time postural techniques to prevent mucous pooling, and hydrating techniques such as a humidifier to keep mucous from hardening. Radiation-induced sialorrhea may result in difficulty swallowing, gagging with reflex vomiting, and altered sleep ­patterns. Generally, sialorrhea abates within 1–3 months treatment. Of note, patients undergoing radiation therapy may experience both xerostomia and sialorrhea. Unfortunately, treatment approaches that improve one symptom may exacerbate the other. Thus, treatment must be tailored to the individual to maximize symptom control. Chronic sialorrhea is more common in the postoperative setting and has been approached using a number of treatment techniques including anticholinergics, botulinum toxin, and salivary gland excision [92].

Trismus Abnormalities in jaw motion resulting in either mal-­occlusion or trismus are a common but frequently overlooked complication of head and neck cancer therapy. Normal occlusion requires the following structures: mandible, maxilla, muscles of mastication (including the pterygoids, masseter, and temporalis), dentition, an intact neurologic supply, and an adequate vascular supply. When the structures function normally, the mandible has six degrees of motion: depression, elevation, protrusion, retraction, and right and left lateral movement. Damage to any of these structures either by tumor or treatment may result in abnormal occlusion and/or decreased range of motion in the jaw. Trismus is defined as a restriction in range of motion of the jaw. While differing criteria have been used to assess and report trismus, most studies report the maximal inter-incisor opening (MIO) measured in millimeter. Although the criteria for mild, moderate, and severe trismus varies, general guidelines are as follows: greater than 40 mm – normal, between 30 and 40 mm – mild trismus, 15–30 mm – moderate trismus, and <15 mm – severe trismus [93]. Radiation-induced trismus is secondary to fibrosis of the muscles of mastication. There is a strong correlation between the radiation dose to the muscles of mastication and subsequent development of alterations in jaw range of motion [94]. The incidence of radiation-induced trismus has not been well established. This is largely due to the variability in measurement techniques and the heterogenous populations

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studied [95]. Rates as high as 45% have been reported in patients receiving curative doses of radiation therapy involving the muscles of mastication and/or the ligaments of the temporomandibular joint [96]. Current data do not demonstrate an increase in the incidence or severity of symptoms with the use of concurrent chemotherapy. Trismus usually begins to develop 1–9 months after the completion of radiation therapy, however, late-onset trimus has been reported [97]. Trismus is usually permanent and may be progressive; thus, once it develops, ongoing supportive measures are required. Trismus is associated with a number of clinically important sequelae that merit close scrutiny. Decrease range of motion in the jaw may lead to alterations in oral intake, and when severe, patients may be limited to a liquid diet. Rigorous oral care, which is vital in patients with radiation-induced xerostomia, may be difficult or impossible. Speaking may be harder and patients may have trouble being understood. It is important to note that oral intubation or dental procedures may not be feasible in patients with severe decrease in jaw range of motion. Treatment options for trismus are limited. Hyperbaric oxygen therapy, pentoxyfilline [98], and botulinum toxin [99] have been investigated as potential therapeutic interventions, however, data is lacking to support any of these methodologies. Physical therapy with stretching of the muscles is commonly recommended. Although patients with cancerrelated trismus do not experience dramatic improvement in jaw range of motion with physical therapy, deterioration may be prevented [100]. Appliances have been developed to maximize stretching of muscles and soft tissues [95].

Mucosal Sensitivity Radiation therapy is associated with a unique posttreatment pain syndrome – Post Radiation Mucosal Sensitivity (PRMS). Characteristically, patients will complain of burning oral or pharyngeal pain that persists after resolution of the visible ulcerative lesions of mucositis. Symptoms are often exacerbated by spicy or hot food, xerostomia, and dry air. Although symptoms may lesson over time, sensitivity may persist long term. PRMS is a neuropathic pain which may be related to peripheral nerve sensitization and up regulations of Na+ channels by mucositis-associated inflammatory [101]. The cornerstone of treatment for PRMS is to avoid foods or environmental conditions that provoke pain. For patients with oral symptoms requiring intervention, topical anesthetics such as lidocaine (Na+ channel blockers) or ketamine (NMDA inhibitors) may be highly effective. If these agents fail or if patients have pharyngeal pain at sites that preclude administration of topical agents, systemic agents may be needed. Opioids may partially alleviate symptoms in some patients, however, PRMS is a neuropathic pain, thus it tends

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to be opioid resistant. When indicated, adjunctive pain ­medication such as clonazepam, gabapentin, and other antidepressants may be tried.

Dermatitis Radiation therapy damages radiosensitive keratinocytes found in the basal layer of the epidermis preventing normal maturation and repopulation. As progenitor cells die during radiation, few cells are left in the germinal layer to replenish the normally desquamating upper epithelium. This results in sloughing of the epidermis, exposing the underlying dermal tissue. Reactions are often worse in skin folds and areas of decreased tissue thickness such as around the pinna or at the laryngeal prominence. The administration of concurrent chemotherapy agents (such as cetuximab [102, 103], doxorubicin, actinomycin D, bleomycin, hydroxyurea, 5-fluorouracil, methotrexate, and taxanes) may increase the incidence and severity of symptoms. Other risk factors for the development of radiation dermatitis include: age, nutritional status, diabetes, and concurrent medications [104, 105]. A number of systems have been used to grade acute radiation dermatitis. The CTCAE 3.0 criteria are as follows: grade 1 – faint erythema or dry desquamation; grade 2 – moderate to brisk erythema or patchy moist desquamation, mostly confined to the skin folds and creases with moderate edema; grade 3 – confluent moist desquamation, ³1.5  cm diameter, not confined to skin folds, with pitting edema; and grade 4 – skin necrosis or ulceration of full thickness dermis, may include bleeding not induced by minor trauma or abrasion. Acute radiation dermatitis usually begins within 2–3 weeks of initiating therapy and worsens over time. Once radiation has completed, the skin lesions resolve rapidly (over 2–3 weeks). Long-term patients may experience hypopigmentation, hyperpigmentation, textural changes, loss of hair follicles, and loss of sebaceous glands [106]. In addition, patient may experience fibrosis of the dermis and subcutaneous tissue leading tissue retraction and decreased range of motion or atrophy with increased skin fragility [106, 107]. Patients must be educated regarding the care of acute and late dermal effects of radiation therapy. The reader is referred to a number of manuscripts that provide thorough recommendations for the management of acute and late dermatitis [106, 108, 109].

Conclusions Head and neck cancer is associated with a number of symptom control and functional issues. Although much attention has been directed at the acute effects of therapy, there is

17  Approaches to Supportive Care

increasing recognition of the importance of late effects. The acute and late effects of therapy span a wide range of clinical issues; thus, they require the expertise of a wide array of practitioners. In order to maximize symptom and functional outcomes, a coordinated multidisciplinary approach is needed. Bringing together a team that is able to care for patients in a holistic and proactive manner is challenging at best. Nonetheless, it is necessary.

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264 37. Murphy BA, Friedman J, Dowling E, Cheatham R, Cmela A. Dietary intake and adaptations in head and neck cancer patients treated with chemoradiation. Proc Am Soc Clin Oncol. 2002; 21:abstract # 932. 38. Pauloski BR et al. Swallow function and perception of dysphagia in patients with head and neck cancer. Head Neck. 2002;24(6):555–65. 39. Cheng S, Terrell JE, Bradford CR, Ronis DL, Fowler KE, Prince ME, et  al. Variables associated with feeding tube placement in head and neck cancer. Arch Otolaryngol Head Neck Surg. 2006;132:655–61. 40. Schueren, M.v.B.-d.v.d. Differences in immune status between well nourished and malnourished head and neck cancer patients. Clin Nutr 1998;17(3):107–11. 41. Brookes GB. Nutritional status-a prognostic indicator in head and neck cancer. Otolaryngol Head Neck Surg. 1985;93:69–74. 42. Schueren, M.v.B.-d.v.d. The impact of nutritional status on the prognoses of patients with advanced head and neck cancer. Cancer. 1999;86:519–27. 43. Newman LA et al. Eating and weight changes following chemoradiation therapy for advanced head and neck cancer. Arch Otolaryngol Head Neck Surg. 1998;124:589–92. 44. Schueren, M.v.B.-d.v.d. The impact of nutritional status on the prognosis of patients with head and neck cancer. Cancer. 1999;86:519–27. 45. Hammerlid E et  al. Malnutrition and food intake in relation to quality of life in head and neck cancer patients. Head Neck. 1998;20:540–8. 46. Colasanto JM, Prasad P, Nash MA, Decker RH, Wilson LD. Nutritional support of patients undergoing radiation therapy for head and neck cancer. Oncology. 2005;19(3):371–87. 47. Minasian A, Dwyer JT. Nutritional implications of dental and swallowing issues in head and neck cancer. Oncology. 1998;12(8):1155–62. 48. Harris JA, Benedict FG. A biometric study of basal metabolism in man. Publication 279. Washington, DC: Carnegie Institution; 1919. p. 40–4. 49. Gibson S, Wenig B. Percutaneous endoscopic gastrostomy in the management of head and neck carcinoma. Laryngoscope. 1992;102:977–81. 50. Mekhail TM et al. Enteral nutrition during the treatment of head and neck carcinoma: is a percutaneous endoscopic gastrostomy tube preferable to a nasogastric tube? Cancer. 2001;91(9): 1785–90. 51. Lin A, Jabbari S, Worden FP, Bradford CR, Chepeha DB, Teknos T, et al. Metabolic abnormalities associated with weight loss during chemoirradiation of head and neck cancer. Int J Radiat Biol Phys. 2005;63:1413–8. 52. Tyldesley S et  al. The use of radiologically placed gastrostomy tubes in head and neck cancer patients receiving radiotherapy. Int J Radiat Oncol Biol Phys. 1996;36(5):1205–9. 53. Beer KT, Krause KB, Zuercher T, Stanga Z. Early percutaneous endoscopic gastrostomy insertion maintains nutritional state in patients with aerodigestive tract cancer. Nutr Cancer. 2005;52(1): 29–34. 54. Bergstrom LR, Larson D, Zinsmeister AR, Sarr MG, Silverstein MD. Utilization and outcomes of surgical gastrostomies and jejunostomies in an era of percutaneous endoscopic gastrostomy: a population-based study. Mayo Clin Proc. 1995;70:829–36. 55. Silver HJ, Wellman NS, Arnold DJ, Livingstone AS, Byers PM. Older adults on home enteral nutrition: enteral regimen, provider involvement, and health care outcomes. J Parenter Enteral Nutr. 2004;28:92–8. 56. Beecken L, Calaman F. A return to “Normal Eating” after curative treatment for oral cancer. Eur J Cancer B Oral Oncol. 1994; 30B:387–92.

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17  Approaches to Supportive Care benefit in irradiated head and neck cancer. Int J Radiat Oncol Biol Phys. 2000;48(4):1035–9. 79. Bardet E, Martin L, Calais G, Alfonsi M, Feham N, Tuchais C, et al. Subcutaneous versus intravenous administration of amifostine for head and neck cancer patients receiving radiotherapy: preliminary results of the GORTEC 2000-02 randomized trial. Int J Radiat Oncol Biol Phys. 2005;63(2):S127. 80. Brosky M. The role of saliva in oral health: strategies for prevention and management of xerostomia. J Support Oncol. 2007; 5:215–25. 81. Scarantino C et al. Effect of pilocarpine during radiation therapy: results of RTOG 97-09, a phase III randomized study in head and neck cancer patients. J Support Oncol. 2006;4(5):252–8. 82. Kam MKM, Leung SF, Zee B, Chau R, Suen JJS, Mo F, et  al. Prospective randomized study of intensity-modulated radiotherapy on salivary gland function in early-stage nasopharyngeal carcinoma patients. J Clin Oncol. 2007;25(31):4873–9. 83. Chao KS et al. Intensity-modulated radiation therapy reduces late salivary toxicity without compromising tumor control in patients with oropharyngeal carcinoma: a comparison with conventional techniques. Radiother Oncol. 2001;61(3):275–80. 84. Daly ME, Lieskovsky Y, Pawlicki T, Yau J, Pinto H, Kaplan M, et al. Evaluation of patterns of failure and subjective salivary function in patients treated with intensity modulated radiotherapy for squamous cell head and neck cancer. Head Neck. 2007;29: 211–20. 85. Saarilahti K, Kouri M, Collan J, Kangasmaki A, Atula T, Joensuu H, et al. Sparing of the submandibular glands by intensity modulated radiotherapy in the treatment of head and neck cancer. Radiat Oncol. 2006;73:320–5. 86. Johnson JT et al. Oral pilocarpine for post-irradiation xerostomia in patients with head and neck cancer. N Engl J Med. 1993; 329:390–5. 87. Chambers MS et al. Cevimeline for the treatment of postirradiation xerostomia in patients with head and neck cancer. Int J Radiat Oncol Biol Phys. 2007;68(4):1102–9. 88. Oral health in cancer therapy: a guide for health care professionals. www.doep.org 89. Keck T, Rozsasi A, Leiacker R, Scheithauer MO. Lower airway humidification in spontaneously breathing tracheostomized patients: comparative study of trachea spray versus heated humidification. Head Neck. 2008;30:582–8. 90. Dreizen S, Brown LR, Daly TE, Drane BE. Prevention of xerostomia-related dental caries in irradiated cancer patients. J Dent Res. 1977;56(2):99–104. 91. Papas A et al. Caries clinical trial of a remineralising toothpaste in radiation patients. Gerodontology. 2008;25(2):76–88. 92. Bomeli SR, Desai S, Johnson JT. Management of salivary flow in head and neck patients. Oral Oncol. 2008;44:1000–8. 93. Dijkstra PU, Huisman PM, Roodenburg JLN. Criteria for trismus in head and neck oncology. Int J Oral Maxillofac Surg. 2006;35:337–42. 94. Teguh DN, Levendag PC, Voet P, van der Est H, Noever I, de Kruijf W, et  al. Trismus in patients with oropharyngeal cancer: relationship with dose in structures of mastication apparatus. Head Neck. 2008;30:622–30. 95. Dijkstra PU, Kalk WWI, Roodenburg JLN. Trismus in head and neck oncology: a systematic review. Oral Oncol. 2004;40(9): 879–89.

265 96. Kent ML, Bennan MT, Noll JL, Fox PC, Burri SH, Hunter JC, et al. Radiation-induced trismus in head and neck cancer patients. Support Care Cancer. 2008;16:305–9. 97. Wang CJ, Huang EY, Hsu HC, Chen HC, Fang FM, Hsiung CY. The degree and time-course assessment of radiation-induced trismus occurring after radiotherapy for nasopharyngeal cancer. Laryngoscope. 2005;115:1458–60. 98. Chua D, Lo C, Yuen J, Foo YC. A pilot study of pentoxifylline in the treatment of radiation-induced trismus. Am J Clin Oncol. 2001;24(4):366–9. 99. Hartl DM, Cohen M, Julieron M, Marandas P, Janot F, Bourhis J. Botulinum toxin for radiation-induced facial pain and trismus. Otolaryngol Head Neck Surg. 2008;138:459–63. 100. Dijkstra PU, Sterken MW, Pater R, Spijkervet FKL, Roodenburg JLN. Exercise therapy for trismus in head and neck cancer. Oral Oncol. 2007;43:389–94. 101. Finnerup NB, Jensen TS. Mechanisms of disease: mechanismbased classification of neuropathic pain – a critical analysis. Nat Clin Pract Neurol. 2006;2(2):107–15. 102. Bonner JA, Ang K. More on severe cutaneous reaction with radiotherapy and cetuximab. N Engl J Med. 2007;357:1872–3. 103. Budach W, Bölke E, Horney B. Severe cutaneous reaction during radiation therapy with concurrent cetuximab. N Engl J Med. 2007;367(21):514–5. 104. Sitton E. Early and late radiation-induced skin alterations part 1: mechanism of skin changes. Oncology Nursing Forum. 1992;19(5):801. 105. Blackmar A. Radiation-induced skin alterations. Medsurg Nursing. 1997;6(3):172–5. 106. Hymes SR, Strom EA, Fife C. Radiation dermatitis: clinical presentation, pathophysiology, and treatment 2006. J Am Acad Dermatol. 2006;54(1):28–46. 107. Stohl R. The nursing role in radiation oncology: symptom management of acute and chronic reactions. Oncol Nurs Forum. 1988;15(4):429–34. 108. D’haessse S, Bate T, Claes S, Boone A, Vanvoorden V, Efficace F. Management of skin reactions during radiotherapy: a study of nursing practice. Eur J Cancer Care. 2005;14:28–42. 109. Bernier J et  al. Consensus guidelines for the management of ­radiation dermatitis and coexisting acne-like rash in patients receiving radiotherapy plus EGFR inhibitors for the treatment of squamous cell carcinoma of the head and neck. Ann Oncol. 2008; 19:142–9. 110. World Health Organization. A handbook for reporting results of cancer treatment. Geneva: World Health Organization; 1979. 111. Trotti A et  al. CTCAE v3.0: development of a comprehensive grading system for the adverse effects of cancer treatment. Semin Radiat Oncol. 2003;13(3):176–81. 112. American Speech-Language-Hearing Association. Roles of speech-language pathologists in swallowing and feeding disorders: technical report [Technical Report]. 2001. Available from www.asha.org. 113. Molseed L. Clinical evaluation of swallowing: the nutritionist’s perspective. In: Murry CA, editor. Comprehensive management of swallowing disorders. San Diego, CA: Plural Publishing; 2006. p. 59. 114. Blackburn GL, Bistrian BR, Miani BS, Schlamm HT, Smith MF. Nutritional and metabolic assessment of the hospitalized patient. J Parenter Enteral Nutr. 1977;1:11–22.

Chapter 18

Intensity-Modulated Radiation Therapy for Head and Neck Cancer Nancy Lee, Daniel Gomez, Edward J. Shin, and K.S. Clifford Chao

Abstract  Intensity-modulated radiation therapy (IMRT) has revolutionized the treatment of head and neck cancer. A general overview of IMRT in the treatment of head and neck cancer is provided, focusing on guidelines for target determination and delineation for the different subsites within the head and neck. General facts, general management, target delineation, and IMRT results of specific anatomic subsites are outlined, including the nasopharynx, the oropharynx, the hypopharynx, the larynx, the oral cavity, and the thyroid are discussed, along with cancer of unknown primary. Keywords  Intensity-modulated radiation therapy • Head and neck cancer • Target determination • Target delineation • Subsites

Introduction Intensity-modulated radiation therapy (IMRT) has revolutionized the treatment of head and neck cancer. Compared with conventional opposed lateral fields that were used to treat these tumors, IMRT has provided comparable, if not better, local control with significantly improved long-term toxicities associated with high doses of radiation therapy. The ability to tightly conform to irregularly shaped tumors while limiting the dose delivered to the surrounding critical structures is the hallmark of IMRT. This advantage is especially seen when tumors are located near critical structures, i.e., the brainstem and optic structures, where there are great limitations in delivering effective therapeutic doses of radiation using conventional radiotherapy techniques. In addition,

N. Lee (*) Radiation Oncology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, Box 22, New York, NY 10021, USA e-mail: [email protected]

because there is minimal organ motion in the head and neck, with the use of proper immobilization the planned dose ­distribution can be delivered with great assurance. The theoretical dosimetric advantage of IMRT has translated clinically into improvement in patient quality of life. Several Phase III trials have now demonstrated the beneficial effects of IMRT when compared with conventional radiotherapy in terms of minimizing late toxicities, and in particular xerostomia. The purpose of this chapter is to provide a general overview of IMRT in the treatment of head and neck cancer, focusing on guidelines for target determination and delineation for the different subsites within the head and neck. Clinical updates will also be presented.

Target Determination and Delineation for Head and Neck Cancer The complexity of the head and neck anatomy requires the treating radiation oncologist to carefully and accurately delineate the target volume prior to initiating IMRT. One must have an understanding of the relationship of the various structures to one another and the patterns of spread from the primary tumor site as well as the nodal drainage. To date, no consensus delineation guidelines other than the N0 nonsurgically violated neck have been published. A guideline regarding the different neck lymph node levels can be found in Table 18.1 [1]. It is important not to use the N0 guideline for node-positive or postoperative cases in which the nodal planes are not as well defined either due to the presence of nodes or surgical violation of tissue planes. A proposal, though not a consensus guideline, for the node-positive neck has been published by Gregoire et al. [2]. The probability of nodal drainage to a specific ipsilateral lymph node level is directly related to the location and stage of the primary tumor. Table  18.2 specifies the likelihood of pathologic lymph node involvement in both the clinically positive and negative neck, by anatomic subsites.

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_18, © Springer Science+Business Media, LLC 2011

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Table 18.1  Lymph node levels Robbins classification level Terminology

Definition

Ia Ib

Submental Submandibular

II

Upper jugular

III

Middle jugular

IV V VI VII

Lower jugular Spinal accessory Anterior compartment Upper mediastinal

Contains submental/submandibular triangles Bounded by the posterior belly of digastric muscle, hyoid bone and the body of mandible Contains upper internal jugular lymph nodes. Extends from level of hyoid bone to skull base Contains middle internal jugular lymph nodes from hyoid bone to cricohyoid membrane Contains lower internal jugular lymph nodes from cricohyoid membrane to clavicle Posterior triangle lymph nodes bounded by trapezius, sternocleidomastoid, clavicle From hyoid bone to suprasternal notch bounded laterally by the carotid sheath Lymph nodes inferior to suprasternal notch in the upper mediastinum

Table 18.2  Incidence and distribution of lymph nodes in N0 and N+ neck Radiologically enlarged retropharyngeal Pathologic nodal metastasis(%) nodes (%) Level I Level II N+

Level III

Level IV

Level V

Clinical presentation

N−

Nasopharynx

40

Oral cavity Oral tongue Floor of mouth Aveolar ridge and RMT

– – –

Oropharynx Base of tongue Tonsil

 0  4

 6 12

 4  0

19  8

30 19

89 74

22 14

22 31

7 9

10 14

0 5

18 12

Hypopharynx Pharyngeal wall Pyriform sinus

16  0

21  9

 0  0

11  2

 9 15

84 77

18  8

72 57

0 0

40 23

0 0

20 22

86 – – –

N−

N+

N−

N+

N−

N+

N−

N+

N−

N+

–

–

–

–

–

–

–

–

–

–

14 16 25

39 72 38

19 12 19

73 51 84

16 7 6

27 29 25

3 2 5

11 11 10

0 0 1

 0  5  4

Larynx Supraglottic larynx  0  4  6  2 18 70 18 48 9 17 2 16 Glottic larynx – –  0  9 21 42 29 71 7 24 7  2 From Chao KSC, Wippold FJ, Ozyigit G, Tran BN, Dempsey JF. Determination and delineation of nodal target volumes for head and neck cancer based on patterns of failure in patients receiving definitive and postoperative IMRT. 2002;53:11. Reprinted with kind permission from Elsevier

General Delineation Guidelines • An excellent reference in the delineation of nodal levels as visualized on computed tomography (CT) slices has been published by the Radiation Therapy Oncology Group (RTOG) (http://www.rtog.org/atlases/hnatlas/ main.html) and the European Organization for Research and Treatment of Cancer (http://groups.eortc.be/radio/ ATLAS.html). • Gregoire et al. [2] has published recommendations for the treatment of the node-positive or postoperative neck. Selected recommendations are as follows: −− Target delineation should include the retrostyloid space up to the skull base when level II is involved. −− Supraclavicular fossa would be included when level IV or Vb is involved.

−− The entire muscle should be included in the target when there is clear extracapsular extension. −− The entire surgical field (“surgical bed”) should be included in the target in postoperative cases. • Extracapsular extension is a significant independent risk factor for local recurrence and distant metastasis. The clinical target volume (CTV) should be extended to the skin to account for microscopic spread. • An “all in one” IMRT technique where all treated regions are being included in the IMRT fields is preferred over “split-field” IMRT when the low neck contains involved lymph nodes, or if the primary tumor is located in the larynx, hypopharynx, and thyroid. A “split-field” technique is preferred in all other scenarios in an attempt to ­minimize the dose delivered to the normal larynx. A low anterior neck field is then matched to the IMRT fields. The ­common match point is just above the arytenoids cartilages, which

18  Intensity-Modulated Radiation Therapy for Head and Neck Cancer

•

•

•

•

•

•

will ensure adequate dosimetric coverage to the level II lymph nodal regions. A “cheater” spinal cord block is placed at the match point, approximately 2 × 2 cm, to add an extra layer of protection over the spinal cord in the region of the match line. The size of the lymph node denotes whether it should be included in the gross target volume (GTV). Lymph nodes with a minimal axial diameter of more than 1.1 cm in the subdigastric region and more than 1.0 cm in other nodal regions is considered suspicious for metastasis. Lymph nodes with a necrotic center should also be considered within the GTV. Communication between the operating surgeon and the radiation oncologist is crucial to ensure adequate delineation of the postoperative case. Imaging studies that are helpful to accurately define the gross extent of disease include CT with contrast, magnetic imaging resonance (MRI) with gadolinium, and positron emission tomography (PET) scans. Nodes that are smaller than 1  cm but are PET avid should be included in the ­target volume as GTV. PET and MRI fusion treatment planning is being used at an increasing number of institutions. While the treating physician should exercise caution in strictly defining the GTV and CTV in correlation with areas of increased fluorodeoxyglucose (FDG) uptake, these more sensitive imaging studies can provide useful information in target delineation. Different CTVs are established for all targets within one plan along with suggested dosing. −− CTV1: highest dose region, margin given to GTV or the postoperative surgical bed. Definitive cases: 70 Gy, postoperative dose: 60–66 Gy. −− CTV2: intermediate dose region, which is at high-risk but clinically uninvolved regions. Definitive cases: 59.4–63 Gy; postoperative dose: 54–60 Gy. −− CTV3: low-dose region including regions at a lower risk for microscopic disease. Definitive cases: 54–56 Gy; Postoperative dose: 54 Gy

Treatment of Specific Anatomic Subsites Nasopharynx General Facts • Anterior border: posterior choanae Posterior border: at the level of the first two cervical ­vertebrae and clivus

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Superior border: basisphenoid, basiocciput Inferior border: soft palate Lateral border: pharyngeal fascia including the eustachian tube. • Approximately 85–90% of patients with nasopharyngeal cancer have lymph node involvement and 50% have bilateral lymph node involvement. Nodal drainage can be direct to level V, through the lateral pharyngeal walls to the retropharyngeal and subdigastric nodes. Therefore, levels I–V are all at risk for involvement. • Anatomic knowledge of the skull base is important as nasopharyngeal tumors can involve multiple cranial nerves including II–VI and IX–XII. • The World Health Organization divides nasopharyngeal carcinoma (NPC) into the following: keratinizing squamous cell carcinoma; nonkeratinizing carcinoma, which subdivides into differentiated and undifferentiated; and basaloid squamous cell carcinoma. Lymphoepithelial carcinoma is a further subtype that represents nonkeratinizing and undifferentiated carcinomas with an abundance of lymphocytes.

General Management • Treatment consists of definitive radiation therapy ± cisplatin followed by adjuvant chemotherapy, though there are debates regarding the added benefit of adjuvant chemotherapy. • The 5-year overall survival rates range from 35 to 60%. • In the Phase III trial (Al-Sarraf et al. [3]), patients with stage III–IV NPC were randomized to radiotherapy alone (70  Gy) or radiotherapy with concurrent cisplatin (100 mg/m2) every 3 weeks during treatment, followed by cisplatin (80 mg/m2) and fluorouracil (1,000 mg/m2/day), 4 days every 4 weeks after the completion of radiation therapy. At 5 years, overall survival was 37% vs. 67% in the radiotherapy alone vs. chemoradiation arms, respectively, and progression-free survival was 29% vs. 58% in the radiotherapy alone vs. chemotherapy arms, respectively. • A more recent Phase III study from Singapore [4] randomized 221 patients to radiation alone (70  Gy in 7 weeks) or concurrent cisplatin (weeks 1, 4, and 7 of radiation, 25 mg/m2), followed by adjuvant cisplatin (20 mg/m2) and fluorouracil (1,000  mg/m2) every 4 weeks for three cycles after the completion of radiation therapy. This trial has a design nearly identical to the US Intergroup Trial. The 3-year overall survival rate was 80% vs. 65% for the chemoradiation vs. the radiation-alone arm, respectively, with a hazard ratio for overall survival of 0.51 (p = 0.0061). This trial confirmed the findings of the Intergroup Trial.

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Table 18.3  Suggested target delineation guidelines for nasopharyngeal cancer Stage CTV1 CTV2 T1–T4N0

GTV + 5–10 mm

Entire nasopharynx, clivus, skull base, pterygoid fossae, parapharyngeal space, sphenoid sinus, posterior 1/4 to 1/3 of maxillary sinus and nasal cavity, bilateral retropharyngeal regions, bilateral levels II–V T1–T4N1–3 GTV + 5–10 mm As above and include bilateral level I At the discretion of the treating physician, the CTV margin can be as small as 1 mm in regions near critical normal tissues, i.e., brain stem

• Several meta-analyses demonstrated that the addition of chemotherapy to radiation therapy increased both progression-free and overall survival. Target Delineation for IMRT • Table  18.3 contains the suggested guidelines for target delineation in NPC. The GTV includes the primary tumor and involved lymph nodes. • Due to the high probability of lymph node metastases, levels IB-V and the retropharyngeal lymph nodes should be included in the CTV bilaterally. Level I can be omitted in N0 cases. CTV also includes areas where NPC is likely to spread: the entire nasopharynx, posterior 1/3 of the nasal cavity and maxillary sinuses, parapharyngeal fat, clivus, and skull base. • Figure 18.1 depicts a sample target volume for a patient with locally advanced NPC. The planning target volume (PTV) represents the final treatment volume, and is the CTV with an “adequate” margin at the physician’s discretion, to account for patient day to day set-up errors as well as organ motion.

• Due to the encouraging locoregional control as well as improved salivary function with IMRT for NPC, the RTOG conducted a Phase II multi-institution trial and the results reproduced the excellent locoregional control rates reported by single institutions, with control rates on the order of 90% [8]. • The predominant failure pattern in patients treated with IMRT for NPC is distant metastasis. Therefore, the RTOG is conducting a Phase II trial (RTOG 0615) in which patients with loco-regionally advanced NPC are being treated with the current standard chemotherapy and IMRT with the addition of the study drug, bevacizumab, a targeted agent directed against the vascular endothelial growth factor, to test whether this addition will further decrease the rate of distant metastasis with the ultimate goal of improving overall survival. The trial is closed to patient accrual and results are pending.

Oropharynx General Facts • The oropharynx consists of four subsites: soft palate, palatine tonsillar region (fossa and pillars), lingual tonsil or base of tongue, and posterior and lateral pharyngeal walls. • The oropharynx has a rich lymphatic network, and ­primarily drains into the subdigastric, upper cervical (II and III), and parapharyngeal lymph nodes (in proximity to cranial nerves IX–XII). Progression of nodal metastases is usually orderly, starting at level II and proceeding inferiorly to levels III and IV. Skip nodal metastases are relatively rare. • The vast majority of tumors of the oropharynx are squamous cell carcinomas.

IMRT Results

General Management

• Two randomized studies on early-stage NPC have demonstrated an advantage of IMRT over conventional techniques in terms of salivary preservation [5, 6]. • Lee et al. [7] reviewed 67 patients who underwent IMRT for NPC at the University of California-San Francisco between 1995 and 2000. At a median follow-up of 31 months, the 4-year locoregional progression-free rate was 98%. Sixteen patients experienced distant metastases. At 24 months, only one of the 41 evaluable patients had Grade 2 xerostomia, with the remaining having Grade 0 or 1 toxicity. Several other single institutions also published similar results.

• Surgery and adjuvant radiation ± chemotherapy was previously the treatment paradigm. • The study RTOG 73-03 (Kramer et al. [9]) was the first to suggest that surgery was not necessary as a component of treatment. This study randomized patients to either ­surgery, preoperative or postoperative radiation therapy or to definitive radiation therapy, reserving surgery for ­salvage treatment. There was no difference in locoregional control or overall survival, and complications were higher in the surgical arms. • Parsons et al. [10] compiled results from 11 institutions from 1970 to 2000 using a MEDLINE search, to determine

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Fig. 18.1  Axial slices of representative slices of a nasopharyngeal carcinoma patient undergoing IMRT

if there was a difference in outcomes for patients treated with surgery ± adjuvant radiation vs. definitive ­radiation ±  neck dissection. While rates of local-regional control, 5-year overall survival, and 5-year cause-specific survival were similar in the two groups, the rate of significant complications was higher in patients who underwent upfront surgery. • Fu et al. [11] performed a randomized trial of over 1,000 patients with locally advanced head and neck cancer, randomizing them to (a) standard fractionation at 2 Gy once daily to 70 Gy, (b) accelerated fractionation, 1.2 Gy BID to 81.6 Gy, (c) accelerated fractionation with a split-course, 1.6 Gy BID to 38.4 Gy, 2-week break, then to 67.2 Gy, or (d) accelerated fractionation with a concomitant boost, 1.8 Gy daily to 72 Gy, with a boost of 1.5 Gy as a second daily treatment for the last 12 fractions. Arms (b) and (d) had better local-regional control than arms (a) and (c). • Denis et al. [12], randomized 226 patients with stage III or IV oropharyngeal carcinoma to either (a) radiation

alone (70  Gy in 2  Gy fractions) or (b) concomitant ­chemoradiation with the regimen above and carboplatin (70  mg/m2) with fluorouracil (600  mg/m2). Five-year overall survival (22% vs. 16%), disease-free survival (27% vs. 15%), and locoregional control (48% vs. 25%) all favored the chemoradiation arm. • Pignon et al. [13] performed a meta-analysis that included trials between 1965 and 2000 of patients with carcinoma of the oropharynx, oral cavity, larynx, or hypopharynx; there was an overall survival benefit of approximately 6.5% in 5 years in favor of concomitant chemoradiotherapy.

Target Delineation • Table 18.4 depicts suggested guidelines for target delineation in oropharyngeal carcinoma. • Note that the bilateral neck is covered in all oropharyngeal lesions other than T1N0 and small well-lateralized

272 Table 18.4  Suggested target delineation guidelines for oropharyngeal cancer

N. Lee et al. Site/stage

CTV1

CTV2

CTV3

Tonsil / T1N0 GTV + 5–10 mm Ipsilateral levels IB-V a, RP Tonsil / T2–T4N0 GTV + 5–10 mm Bilateral levels IB-V a RP Tonsil / T1–T4N+ GTV + 5–10 mm Ipsilateral IB-V RP Contralateral Ib-V, RP Base of Tongue Soft GTV + 5–10 mm Bilateral IB-V a, RP Palate T1–T4N0 Base of Tongue Soft GTV+ 5–10 mm Ipsilateral Ib-V, RP Contralateral Ib-V a, RP Palate T1–T4N+ Note: For all dosing, the treating physician can also decide on whether the N0 nodal CTVs are treated with the CTV2 or CTV3 dose RP retropharyngeal nodes a  At the discretion of the treating physician, can treat levels II–IV in N0 neck  

Fig. 18.2  Axial slices of representative slices of a oropharyngeal carcinoma patient undergoing IMRT

T2N0 tonsillar lesions without soft palate or base of tongue involvement. • Figure 18.2 depicts the delineation of a representative patient from Memorial Sloan-Kettering Cancer Center (MSKCC).

IMRT Results • Chao et al. [14] reviewed 74 patients with squamous cell carcinoma of the oropharynx (all stages) treated with IMRT. Thirty-one received definitive IMRT and the remaining were treated postoperatively. Four-year overall survival and disease-free survival were 87 and 81%, respectively. Fifteen patients experienced Grade 3 or higher skin toxicity, while 32 experienced Grade 3 or higher mucosal toxicity (28 with Grade 3). There were no Grade 3 or higher late toxicities. The most common late toxicity was xerostomia; there were 32 patients with Grade 1 and nine patients with Grade 2 late toxicity. • In a study by de Arruda et al. [15] at MSKCC, 50 patients with oropharyngeal carcinoma treated with IMRT between

1998 and 2004 were analyzed (78% stage IV disease, 96% with definitive treatment). Two-year local control and overall survival were both 98%. Thirty-one patients had Grade 3 acute toxicities, none had Grade 4 acute toxicities. 67% had Grade 0–1 late toxicities, and the remainder had Grade 2 late toxicities. Of the 42 patients that had a percutaneous endoscopic gastrostomy (PEG) tube placed at the beginning of treatment, 36 had the PEG tube removed at the time of analysis.

Hypopharynx General Facts • The anatomical boundaries of the hypopharynx are as ­follows: superior, hyoid bone; inferior, inferior edge of cricoid cartilage. The pyriform sinuses are lateral to the vocal cords, but the apices of the pyriform sinuses extend inferiorly to the vocal cords.

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• Superior to the hypopharynx is the oropharynx, and inferiorly lies the most superior portion of the esophagus (the cervical esophagus). • There is significant lymphatic drainage to the hypopharynx. Three main pathways exist: (1) through the internal branch of the superior laryngeal artery to levels II and III, (2) through the paratracheal lymph nodes into level IV and the mediastinal lymph nodes, and (3) to the retropharyngeal lymph nodes. • The most common site of lymph node metastasis is to level II. • Almost all hypopharyngeal tumors are squamous cell carcinomas.

General Management • T1–T2N0 disease can be treated with either definitive radiation or surgery. • Conservative surgery for early-stage disease entails a ­partial laryngopharyngectomy with ipsilateral neck dissection. Patients with N2C disease undergo a bilateral neck dissection. • The following are contraindications for conservation ­surgery: vocal cord paralysis, pyriform sinus apex invasion, cartilage invasion, extralaryngeal extension, and/or arytenoid involvement. • For locally advanced disease, including T3–T4 or nodepositive tumors, surgery with adjuvant radiation ± chemotherapy or concurrent chemoradiotherapy is the treatment of choice. • The surgery for locally advanced disease is a total laryngectomy and partial pharyngectomy with neck dissection. • Multiple retrospective studies have demonstrated the ­efficacy of postoperative radiation therapy for advanced tumors [16–18]. • Randomized studies have shown the added benefit of ­chemotherapy given concurrently with postoperative radiation therapy in patients with high-risk features, i.e., positive margins or extracapsular extension [19–21]. • In a Phase III trial by Lefebvre et al. [22], patients with T2–T4N0-N2b disease were assigned to either: (a) immediate laryngectomy with postoperative radiotherapy (50–70 Gy) or (b) induction chemotherapy with cisplatin (100 mg/m2) and fluorouracil infusion (1,000  mg/m2), followed by either radiation (70  Gy) in the responders or laryngectomy followed by postoperative radiation (50–70 Gy) in the nonresponders. While local failures were approximately the same in the two arms (12% vs. 17%), there were fewer distant failures in arm b (25% vs. 36%), and the median overall survival was also greater (44 months vs. 25 months). The authors concluded that laryngeal

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p­ reservation is a feasible approach in patients with locally advanced hypopharyngeal cancer. • Several randomized trials comparing chemoradiotherapy to radiotherapy alone included hypopharyngeal carcinoma and have shown improved locoregional control, disease-free survival, and overall survival in the combined-modality arm. Target Delineation • Table  18.5 depicts suggested target volumes for patients with hypopharyngeal tumors. GTV includes all gross ­disease and any clinically involved lymph nodes. • Due to the high likelihood of lymphatic spread, levels II–V should be included in the field along with retropharyngeal nodal regions. Please see Table  18.5 for further details. • Figure 18.3 depicts representative CT slices from a patient with locally advanced hypopharyngeal carcinoma.

IMRT Results • Lee et al. [23] analyzed 20 patients with laryngeal cancer and 11 patients with hypopharyngeal cancer treated with IMRT and concurrent platinum-based chemotherapy at MSKCC, most of whom had stage IV disease. Two-year locoregional control for the patients with hypopharyngeal tumors was 73%, and 2-year overall survival was 53%. Four of the eleven pati-ents were PEG-tube dependent at the time of the ­analysis, and the 2-year PEG-tube dependency rate was 31%.

Larynx General Facts • The larynx is divided into three subsites: the supraglottis, the glottis, and the subglottis. • The supraglottis contains the following: epiglottis, ­aryepiglottic folds, arytenoids, and false vocal cords. Table 18.5  Suggested target delineation guidelines for hypopharyngeal cancer Site/stage CTV1 CTV2 CTV3 T1–T4N0

GTV + 5–10 mm

T1–T4N+

GTV + 5–10 mm

Bilateral levels II–V a, RP Ipsilateral Contralateral levels I–V, RP levels II–V, RP

RP retropharyngeal nodes At the discretion of the treating physician, can treat levels II–IV in N0 neck a

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Fig. 18.3  Axial slices of representative slices of a hypopharyngeal carcinoma patient undergoing IMRT

•

•

• •

The supraglottis has a significant amount of lymphatic drainage. Through the thyrohyoid membrane, the lymphatic drainage proceeds to levels II–IV. The glottis contains the true vocal cords and the anterior and posterior commissures. There are no lymph nodes that drain from the true vocal cords. Lymph node metastases from tumors of the true vocal cords occur with extension of the tumor to the subglottis or supraglottis. The subglottis extends from the lower boundary of the glottis to the inferior aspect of the cricoid cartilage. The subglottis drains to prelaryngeal, lower jugular, pretracheal, and upper mediastinal lymph nodes. Greater than 95% of laryngeal tumors are squamous cell carcinomas. One distinct entity of squamous cell carcinoma in laryngeal cancer is verrucous carcinoma, which is well differentiated and exophytic. It has been cited in the past that these tumors undergo transformation to an aggressive phenotype after radiation, but whether or not this truly occurs remains unclear.

General Management • Carcinoma in situ of the vocal cord can be managed by either radiation therapy, local excision, or laser therapy. With vocal cord “stripping” or laser excision, tumors often recur, and such patients should be referred for radiation therapy. Control rates are above 95% with radiation. • For early-stage carcinoma of the vocal cord (T1–T2N0M0), surgical excision and radiation therapy have been shown to have comparable results. However, voice quality is generally better preserved with radiation therapy. The typical dose is 2.25 Gy to a total dose of 63 Gy for T1 and 65.25 Gy for T2 lesions. • To study locally advanced laryngeal cancer, RTOG 9111 [24] randomized 547 patients with stage III or IV laryngeal carcinoma (T1 tumors and large-volume stage IV excluded) to either (a) induction chemotherapy with ­cisplatin (100  mg/m2) and fluorouracil (1,000  mg/m2) ­followed by radiation therapy (70 Gy in 2 Gy fractions), (b) concurrent radiation (70  Gy in 2  Gy fractions) and

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•

•

•

•

­cisplatin (100 mg/m2 on days 1, 22, and 43), or (c) radiation alone (70  Gy in 2  Gy fractions). The study found that ­concurrent chemoradiation provided an increased rate of larynx preservation at 2 years (88% vs. 75% and 70% in arms b vs. arms a and c, respectively), as well as improved disease-free survival. Early exophytic lesions of the supraglottis (T1N0) can be treated with either definitive radiation or hemilaryngectomy (supraglottic laryngectomy), which provides voice preservation. For intermediate disease (T2NX), definitive chemoradiation and supraglottic laryngectomy offer similar rates of local control. The following are contraindications to supraglottic laryngectomy: bilateral arytenoid involvement, arytenoid fixation, base of tongue involvement, invasion of the thyroid or cricoid cartilage, involvement of the postcricoid region, impaired vocal cord mobility, glottic extension, and/or patients at increased risk of aspiration (elderly, patients with lung disease). For extensive lesions (T3–T4), either voice preservation with chemoradiation or surgery and postoperative radiation ± chemotherapy are utilized. Note that patients with significant thyroid cartilage invasion are usually referred for surgery. Postoperative chemotherapy should be considered in patients with a positive margin or extracapsular extension. Subglottic tumors are rare and are usually diagnosed at an advanced stage. The treatment of choice is typically ­surgery followed by radiation ± chemotherapy. Alternative treatment consists of concurrent chemoradiotherapy.

Target Delineation • Table  18.6 demonstrates the suggested target delineation for a patient with supraglottic cancer. As noted above, subglottic tumors are rare and treatment should be individualized depending on the clinical situation. • Laryngeal cancer (other than T1–T2N0 glottic tumors) is generally treated using an “all-in-one” technique. No low anterior neck field is utilized. • As noted above, in T1–T2N0 tumors the neck is generally not treated. However, in T2N0 tumors that are bulky, or with subglottic extension, the physician can consider treating the bilateral neck, as described for T3–T4N0 tumors.

IMRT Results • In the Lee et al. [23] study cited above, 20 patients with laryngeal cancer (and mainly stage IV disease) were treated with IMRT and concurrent platinum-based ­chemotherapy. The 2-year rates of locoregional control and overall ­survival

275 Table  18.6  Suggested target delineation guidelines for laryngeal cancer Site/stage CTV1 CTV2 CTV3 Supraglottic T1–T4N0 GTV + 5–10 mm T1–T4N+

GTV + 5–10 mm

Glottic T3–T4N0

GTV + 5–10 mm

T1–T4N+

GTV + 5–10 mm

Bilateral levels II–V a, RP Ipsilateral Contralateral levels I–V, RP levels II–V, RP Bilateral levels II–V a Ipsilateral levels I–V

Contralateral levels II–Va, RP Note: RP nodal regions should be covered if there is involvement of the hypopharynx or there are involved cervical lymph nodes RP retropharyngeal nodes a  At the discretion of the treating physician, can treat levels II–IV in N0 neck

were 90 and 69%, respectively, for the patients with laryngeal cancer. One patient developed laryngeal necrosis and one patient had an unusual complication of necrotizing fasciitis. The 2-year PEG-tube dependency rate was 15%.

Oral Cavity General Facts • The oral cavity is made up of the lips, ­buccal mucosa, the floor of the mouth, the upper and lower ­gingiva, the anterior two-thirds of the oral tongue, the hard palate, and the retromolar trigone. • The upper lips are drained primarily by level IB (submandibular) lymph nodes, and less commonly by the periauricular and parotid lymph nodes. • The lymphatic drainage to the buccal mucosa is primarily to levels IB and II. • The primary lymphatic drainage of the floor of mouth is to levels IA and II. • The primary lymphatic drainage of the upper gingival is to levels IB and II. • The muscles of the oral tongue are innervated by the hypoglossal nerve, and sensory innervation is through the lingual nerve, which is part of the mandibular branch of the trigeminal nerve (V). Taste sensation is provided by cranial nerve VII. The three most common routes of lymphatic drainage are to levels IB, II, and, less commonly, IA. However, there is also a direct route to level III, and occasionally isolated metastases are found in this region. • The most common lymphatic metastases of the hard ­palate are to levels IB and II.

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• The retromolar trigone primarily drains to levels IB and II. • Squamous cell carcinoma accounts for the vast majority of cases.

General Management • Definitive surgery is the preferred treatment of choice for all oral cavity cancers unless there is a contraindication. Postoperative radiation therapy is given to those at high risk for recurrence. • Chemotherapy has been shown to benefit patients with positive margins or extracapsular extension, as detailed above in the Cooper et al. and Bernier et al. studies [19–21].

Target Delineation • Due to the higher propensity for oral cavity tumors (and in particular floor of mouth and oral tongue cancer) to invade lymph node level I, these lymph nodes should be included in the neck volumes. Therefore, in the positive neck, levels I–V should be included. In the node negative contralateral neck, levels I–IV should be included. • Coverage for the postoperative bed should be generous as this anatomic site has been surgically violated. This volume should at least include the preoperative GTV.

Fig. 18.4  Axial slices of representative slices of an oral cavity patient undergoing IMRT

N. Lee et al.

• One can consider sparing the contralateral neck in earlystage lesions of the buccal mucosa, retromolar trigone, and gingiva; for lesions that are not well lateralized the bilateral neck should be treated. • The risk of metastasis to retropharyngeal lymph nodes is low, but these lymph nodes can be treated in locally advanced or midline lesions at the physician’s discretion. • Figure 18.4 demonstrates representative CT slices from a patient with oral tongue cancer treated at MSKCC.

IMRT Results • Yao et  al. [25] recently reported on 55 patients with squamous cell carcinoma of the oral cavity, 91% of whom had stage III or IV disease. At a median follow-up of 17 months, 2-year disease-free and overall survival rates were 82 and 68%, respectively. When examining prognostic factors for locoregional control, the study found that anatomic subsite was predictive, with 2-year rates of locoregional control being 69% in oral tongue cancer, 100% for floor of mouth cancer, and 83% for all other groups together. Extracapsular extension was also found to significantly affect locoregional control. • Studer et  al. [26] analyzed 58 patients with oral cavity cancer treated at the University of Zurich. Twenty-eight of these patients were referred for postoperative treatment, and the remainder for definitive treatment. Forty

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patients had T3 or T4 lesions. Patients treated postoperatively had a 92% rate of local control at 2 years, while those treated with radiation alone had a local control rate of 30–40%. • Gomez et  al. [27] reported a series of 35 oral cavity patients treated with IMRT ± chemotherapy after definitive surgical resection. All patients had stage III–IV disease. With a median follow-up of 28.1 months, the 2 and 3 year estimates of locoregional progression-free survival were 84 and 77%, respectively. The overall survival was 74%. Late complications included trismus (17%) and osteoradionecrosis (5%).

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General Management • The mainstay of management for thyroid carcinoma is surgery. Depending on the extent of disease, this resection can entail a near-total thyroidectomy, total thyroidectomy, or wide composite resection to include the surrounding infiltrated tissue. • External beam radiotherapy is given in select cases where patients are at high risk for local recurrence due to their locally aggressive nature, aggressive histology, or unsatisfactory surgery.

Target Delineation

Thyroid General Facts • The thyroid gland is made up of two lobes. They are joined by the thyroid isthmus. The gland lies posterior to the strap muscles and anterior to the prevertebral muscles, inferior to the thyroid cartilage and with the isthmus overlying the second and third tracheal rings. • The thyroid gland has a rich vascular and lymphatic supply. The lymphatic drainage is primarily to the surrounding lymph nodes of the trachea and esophagus (level VI), with a secondary route being to the cervical lymph nodes, levels I–V. There is also lymphatic ­drainage to level VII.

Fig. 18.5  Axial slices of representative slices of a thyroid cancer patient undergoing IMRT

• The CTV includes the thyroid bed, tracheo-esophageal groove, central compartment, levels II–VII, and the upper mediastinum to the level of the carina. • Figure 18.5 demonstrates representative CT slices from a patient with thyroid cancer treated with IMRT.

IMRT Results • Rosenbluth et al. [28] examined 20 patients with nonanaplastic thyroid carcinoma treated with IMRT. Seventeen of these patients had T4 disease and 16 patients had N1 disease. The median total radiation dose was 63  Gy

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(“high-risk” PTV with a total dose of 59.4–63 Gy, ­positive margins treated to 63–66  Gy). The 2-year local control rate was 85% and the 2-year overall survival rate was 60%. Four of the six deaths were due to metastatic disease. • In terms of toxicity, 7 of 20 patients had Grade 3 acute mucositis, 3 of 20 patients developed Grade 3 pharyngitis, and 2 of 20 patients had Grade 3 skin toxicity. There was no Grade 3 or higher xerostomia.

Cancer of Unknown Primary General Facts • The most commonly involved lymph nodes in cancer of unknown primary (CUP) of the head and neck are levels II and III. Levels I, IV, and V are less commonly involved. • The most common primary site for CUP is the oropharynx, which accounts for approximately 80% of tumors. • The most common histology of CUP is squamous cell carcinoma, with lymphoma, adenocarcinoma, and poorly differentiated tumors being less common. • Multiple studies have examined the role of PET scan in detecting the primary tumor, particularly when conventional techniques have not elucidated the origin of disease. General Management • Patients with N1 disease can be treated with a neck dissection alone if there is no extracapsular extension. However, a review by the Danish Society for Head and Neck Oncology, showed that patients treated with surgery alone had an emerging primary rate of 54% at 5 years and a neck control rate of 58% [29]. • Radiation therapy alone is also an option for patients in lieu of neck dissection. In the same study by the Danish Society, the mucosal control rate was 84% in patients receiving radiation alone and the neck control rate was 50%. • Surgery in combination with radiation therapy has appeared to produce the lowest rates of mucosal primary emergence and neck control. The emerging primary rate in the study above for patients receiving surgery with radiation therapy was 15%. • Patients are usually treated with a field that encompasses the bilateral cervical lymph nodes, the retropharyngeal lymph nodes, and the comprehensive mucosal membranes. However, studies have also been done that utilized ipsilateral neck radiation, particularly for patients with poorer performance status.

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Target Delineation • In addition to lymph node coverage, the mucosal surfaces throughout the head and neck should also be targeted, including the nasopharynx, oropharynx, larynx, and hypopharynx, while the oral cavity is excluded. • The dosing of the different mucosal sites can differ depending on the likelihood of emergence of primary in that site. For example, a patient with Asian descent should receive a higher total radiation dose to the nasopharynx while a Caucasian is more likely to have disease involving the oropharynx and hence a higher total dose should be delivered to that site. • There are situations at the discretion of the treating physician where only the involved neck needs to be treated.

IMRT Results • Klem et al. [30] examined 21 patients treated with IMRT. Fourteen were treated with chemoradiation, and five patients received radiation with definitive intent (rather than in the adjuvant setting). Two-year rates of locoregional survival, distant-metastasis-free survival, and overall survival were 90, 90, and 85%, respectively. • In terms of toxicity, at 6 months posttreatment one patient had greater than Grade 1 xerostomia, and Grade 3 acute skin and mucosal toxicity were 5 and 14%, respectively. PEG tube placement was required in 13 patients, but at last follow-up only one patient was PEG-tube dependent. Three patients experienced esophageal strictures, and all had improvement with dilation.

Conclusions IMRT has resulted in clinical improvement quality of life for patients with head and neck cancer. Yet target delineation remains a challenge, due to the complexity of the head and neck anatomy. Improved imaging promises to help improve the delineation of the extent gross disease, but understanding the patterns of spread of disease from the primary tumor site and the nodal drainage is required.

References 1. Gregoire V, Levendag P, Ang KK, Bernier J, et  al. CT-based ­delineation of lymph node levels and related CTVs in the nodenegative neck: DAHANCA, EORTC, GORTEC, NCIC, RTOG consensus guidelines. Radiother Oncol. 2003;69:227–36.

18  Intensity-Modulated Radiation Therapy for Head and Neck Cancer 2. Gregoire V, Eisbruch A, Hamoir M, Levendag P. Proposal for the delineation of the nodal CTV in the node-positive and the postoperative neck. Radiother Oncol. 2006;79:15–20. 3. Al-Sarraf M, LeBlanc M, Giri PG, Fu KK, et al. Chemoradiotherapy versus radiotherapy in patients with advanced nasopharyngeal cancer: phase III randomized Intergroup study 0099. J Clin Oncol. 1998;16:1310–7. 4. Wee J, Tan EH, Tai BC, Wong HB, et al. Randomized trial of radiotherapy versus concurrent chemoradiotherapy followed by adjuvant chemotherapy in patients with American Joint Committee on Cancer/ International Union against cancer stage III and IV nasopharyngeal cancer of the endemic variety. J Clin Oncol. 2005;23:6730–8. 5. Kam MK, Leung SF, Zee B, Chau RM, et al. Prospective randomized study of intensity-modulated radiotherapy on salivary gland function in early-stage nasopharyngeal carcinoma patients. J Clin Oncol. 2007;25:4873–9. 6. Pow EH, Kwong DL, McMillan AS, Wong MC, Sham JS, Leung LH, et al. Xerostomia and quality of life after intensity-modulated radiotherapy vs. conventional radiotherapy for early-stage nasopharyngeal carcinoma: initial report on a randomized controlled clinical trial. Int J Radiat Oncol Biol Phys. 2006;66:981–91. 7. Lee N, Xia P, Quivey JM, Sultanem K, et al. Intensity-modulated radiotherapy in the treatment of nasopharyngeal carcinoma: an update of the UCSF experience. Int J Radiat Oncol Biol Phys. 2002;53:12–22. 8. Lee N, Harris J, Garden AS, Straube W, et al. Intensity-modulated radiation therapy with or without chemotherapy for nasopharyngeal carcinoma: radiation therapy oncology group phase II trial 0225. J Clin Oncol. 2009;27:3684–90. 9. Kramer S, Gelber RD, Snow JB, Marcial VA, Lowry LD, Davis LW, et al. Combined radiation therapy and surgery in the management of advanced head and neck cancer: final report of study 73-03 of the Radiation Therapy Oncology Group. Head Neck Surg. 1987; 10:19–30. 10. Parsons JT, Mendenhall WM, Stringer SP, Amdur RJ, et  al. Squamous cell carcinoma of the oropharynx: surgery, radiation therapy, or both. Cancer. 2002;94:2967–80. 11. Fu KK, Pajak TF, Trotti A, Jones CU, et al. A Radiation Therapy Oncology Group (RTOG) phase III randomized study to compare hyperfractionation and two variants of accelerated fractionation to standard fractionation radiotherapy for head and neck squamous cell carcinomas: first report of RTOG 9003. Int J Radiat Oncol Biol Phys. 2000;48:7–16. 12. Denis F, Garaud P, Bardet E, Alfonsi M, et al. Final results of the 94-01 French Head and Neck Oncology and Radiotherapy Group randomized trial comparing radiotherapy alone with concomitant radiochemotherapy in advanced-stage oropharynx carcinoma. J Clin Oncol. 2004;22:69–76. 13. Pignon JP, le Maitre A, Maillard E, Bourhis J. Meta-analysis of chemotherapy in head and neck cancer (MACH-NC): an update on 93 randomised trials and 17, 346 patients. Radiother Oncol. 2009;92:4–14. 14. Chao KS, Ozyigit G, Blanco AI, Thorstad WL, Deasy JO, Haughey BH, et al. Intensity-modulated radiation therapy for oropharyngeal carcinoma: impact of tumor volume. Int J Radiat Oncol Biol Phys. 2004;59:43–50. 15. de Arruda FF, Puri DR, Zhung J, Narayana A, et  al. Intensitymodulated radiation therapy for the treatment of oropharyngeal

279 carcinoma: the Memorial Sloan-Kettering Cancer Center ­experience. Int J Radiat Oncol Biol Phys. 2006;64:363–73. 16. Driscoll WG, Nagorsky MJ, Cantrell RW, Johns ME. Carcinoma of the pyriform sinus: analysis of 102 cases. Laryngoscope. 1983; 93:556–60. 17. Frank JL, Garb JL, Kay S, McClish DK, et al. Postoperative radiotherapy improves survival in squamous cell carcinoma of the hypopharynx. Am J Surg. 1994;168:476–80. 18. Slotman BJ, Kralendonk JH, Snow GB, Tiwari RM, Karim AB. Surgery and postoperative radiotherapy and radiotherapy alone in T3-T4 cancers of the pyriform sinus. Treatment results and patterns of failure. Acta Oncol. 1994;33:55–60. 19. Bernier J, Cooper JS, Pajak TF, van Glabbeke M, et al. Defining risk levels in locally advanced head and neck cancers: a comparative analysis of concurrent postoperative radiation plus chemotherapy trials of the EORTC (#22931) and RTOG (# 9501). Head Neck. 2005;27:843–50. 20. Bernier J, Domenge C, Ozsahin M, Matuszewska K, et  al. Postoperative irradiation with or without concomitant chemotherapy for locally advanced head and neck cancer. N Engl J Med. 2004;350:1945–52. 21. Cooper JS, Pajak TF, Forastiere AA, Jacobs J, et  al. Postoperative concurrent radiotherapy and chemotherapy for high-risk squamous-cell carcinoma of the head and neck. N Engl J Med. 2004;350:1937–44. 22. Lefebvre JL, Chevalier D, Luboinski B, Kirkpatrick A, Collette L, Sahmoud T. Larynx preservation in pyriform sinus cancer: preliminary results of a European Organization for Research and Treatment of Cancer phase III trial. EORTC Head and Neck Cancer Cooperative Group. J Natl Cancer Inst. 1996;88:890–9. 23. Lee NY, O'Meara W, Chan K, Della-Bianca C, et  al. Concurrent chemotherapy and intensity-modulated radiotherapy for locoregionally advanced laryngeal and hypopharyngeal cancers. Int J Radiat Oncol Biol Phys. 2007;69:459–68. 24. Forastiere AA, Goepfert H, Maor M, Pajak TF, et  al. Concurrent chemotherapy and radiotherapy for organ preservation in advanced laryngeal cancer. N Engl J Med. 2003;349:2091–8. 25. Yao M, Chang K, Funk GF, Lu H, Tan H, Wacha J, et al. The failure patterns of oral cavity squamous cell carcinoma after intensitymodulated radiotherapy-the University of Iowa experience. Int J Radiat Oncol Biol Phys. 2007;67:1332–41. 26. Studer G, Zwahlen RA, Graetz KW, Davis BJ, Glanzmann C. IMRT in oral cavity cancer. Radiat Oncol. 2007;2:16. 27. Gomez DR, Zhung JE, Gomez J, Chan K, et al. Intensity-modulated radiotherapy in postoperative treatment of oral cavity cancers. Int J Radiat Oncol Biol Phys. 2009;73:1096–103. 28. Rosenbluth BD, Serrano V, Happersett L, Shaha AR, et al. Intensitymodulated radiation therapy for the treatment of nonanaplastic thyroid cancer. Int J Radiat Oncol Biol Phys. 2005;63:1419–26. 29. Grau C, Johansen LV, Jakobsen J, Geertsen P, Andersen E, Jensen BB. Cervical lymph node metastases from unknown primary tumours. Results from a national survey by the Danish Society for Head and Neck Oncology. Radiother Oncol. 2000;55:121–9. 30. Klem ML, Mechalakos JG, Wolden SL, Zelefsky MJ, et al. Intensitymodulated radiotherapy for head and neck cancer of unknown primary: toxicity and preliminary efficacy. Int J Radiat Oncol Biol Phys. 2008;70:1100–7.

Chapter 19

Principles of Systemic Chemotherapy for Squamous Cell Head and Neck Cancer Cristina P. Rodriguez and David J. Adelstein

Abstract  Head and neck squamous cell carcinomas are a group of malignancies that are sensitive to systemic therapy, in part due to the complexity of the molecular aberrations in these malignancies that impair DNA repair mechanisms. Administration of chemotherapy in the treatment of head and neck cancers is guided by treatment goals and patient factors unique to this patient population. The known radiation sensitizing properties of chemotherapy and its ability to impact rates of distant failure have established concurrent chemoradiation as a standard definitive and adjuvant therapy for locally advanced disease. Although known to produce tumor responses, chemotherapy given in the metastatic setting has not been consistently demonstrated to improve overall survival. The combination of chemotherapy with targeted monoclonal antibodies has shown promising results. Future investigation of the role of nonoperative treatments in this disease will likely focus on efforts to decrease late treatment-induced morbidity, exploration of reirradiation with concurrent chemotherapy as a salvage therapy, and further integration of chemotherapy, radiation and targeted therapies in both definitive and palliative management. Keywords  Systemic chemotherapy • Multimodality therapy • Concurrent chemoradiation • Palliative chemotherapy

Introduction Historically, the use of systemic treatments in squamous cell head and neck cancer has required an entirely different approach that taken by the radiation therapist and ­surgeon. For the medical oncologist, the anatomic distinctions so ­critical for locoregional disease management are of ­considerably

D.J. Adelstein (*) Department of Solid Tumor Oncology, Cleveland Clinic Taussig Cancer Center, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Desk R-35, 9500 Euclid Avenue, Cleveland, OH 44195, USA e-mail: [email protected]

less importance than the commonalities that head and neck cancers share. These include the common risk factors of tobacco and alcohol abuse, and the associated comorbidity. In addition, these tumors are histologically similar and tend to be locoregionally aggressive with only a limited metastatic potential. The most important similarity, however, has been the relatively uniform response of head and neck cancers to systemic chemotherapy. Indeed, previously untreated squamous cell head and neck cancer is remarkably sensitive to systemic treatments, particularly when compared to most other common solid tumors [1].

Oncogenesis and the Progression from Benign to Malignant Epithelium The complex process that transforms normal epithelium to invasive squamous cell carcinoma is incompletely understood, and the intense scientific inquiry focused on these events has paved the way for development of effective ­systemic agents for this disease. Malignant transformation is a multistep process that is thought to involve an accumulation of genetic defects and interplay between carcinogen exposure, genetic predisposition, and more recently, viral infection. Tobacco and alcohol are well-established risk factors for head and neck cancer. “Field cancerization” is used to describe the predisposition to malignant transformation along the entire upper aerodigestive tract epithelium as a result of carcinogen exposure [2]. Molecular abnormalities known to occur early in oncogenesis are often observed not only in the premalignant lesions themselves, but the surrounding normal epithelium. Synchronous premalignant and malignant lesions in different areas of the aerodigestive tract have been noted to harbor similar molecular abnormalities. This process is felt to be responsible for the clinical observation of second or third primary upper aerodigestive tract malignancies in patients with heavy alcohol and tobacco exposure successfully treated for their index head and neck squamous cell cancer [3].

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_19, © Springer Science+Business Media, LLC 2011

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The stepwise progression to malignancy is somewhat similar to the colon cancer model of carcinogenesis. One of the first observations supporting this was the reproducible cytogenetic abnormalities identified in hyperplasia, dysplasia, carcinoma in situ, and invasive malignancy [4, 5]. For instance, loss of heterozygosity at the 3p and 9p loci have been frequently observed in early premalignant hyperplastic head and neck mucosal lesions. The transition from hyperplastic to dysplastic epithelium is often characterized by loss of heterozygosity at 17p, and gains in the 11q23 region. With more sophisticated molecular techniques, these chromosomal changes have been found to correspond to genes that play critical roles in cell cycle regulation, specifically the tumor suppressor genes p53, Rb, p16, and cyclin D1. It is becoming increasingly apparent that neoplastic transformation is mediated by a far more complex interaction of factors than genetic mutations in proteins regulating the cell cycle. Gene silencing through epigenetic phenomena, such as hypermethylation of promoter regions of tumor suppressor genes, has been observed [6]. The role of overexpressed cell surface receptors such as EGFR and its downstream signaling cascade mediating cellular immortalization and invasion has been recognized [7]. The influence of genes and proteins responsible for cellular adhesion, such as E-cadherin [8], and matrix metalloproteinases [9], has also been implicated. These more recently identified pathways represent therapeutic targets and avenues for drug development [10]. The role of viral infection in carcinogenesis in head and neck cancer was first recognized in nasopharyngeal cancer. Virtually all cases of endemic undifferentiated nasopharyngeal carcinoma are found to harbor the Epstein–Barr virus. The viral proteins LMP1 and LMP2a are thought to exert transforming effects through intracellular signaling cascades promoting cellular immortalization [11]. These cancers behave differently from head and neck cancer of other subsites, with a predilection for early distant spread but otherwise superior treatment outcomes after therapy for local disease. There has also been increasing recent awareness of a distinct patient population with oropharyngeal cancer harboring high-risk human papillomavirus (HPV) subtypes [12]. These patients may not have a prior exposure to tobacco and alcohol, an observation that has challenged the applicability of the field cancerization theory and the multistep carcinogenesis model to all head and neck cancers. These HPVassociated tumors often contain wild-type p53 and Rb, which are functionally inactivated by viral proteins [13]. Not only are these HPV-positive tumors molecularly distinct, but they also appear to have clinically distinct behavior, and a significantly better prognosis after treatment. Investigation into the optimal therapeutic approach for this unique subset is ongoing.

C.P. Rodriguez and D.J. Adelstein

Treatment Goals and Efficacy Endpoints When defining the management for any patient with cancer, it is critical that a clear treatment goal be identified. If the treatment goal is cure, considerable short- and long-term treatment-induced morbidity may be considered acceptable. Aggressive treatment approaches may still be justified when survival prolongation is possible, even if the disease cannot be cured. When the patient can only be palliated, however, considerable discretion must be exercised in the choice of treatment, and the toxicity considered acceptable. Thus the risk/benefit ratio varies considerably depending on the goal of the treatment and the anticipated outcome. What might be considered to be acceptable risk and toxicity for a potentially curable patient may be entirely unacceptable for a patient treated with palliative intent. Multiple efficacy endpoints are used in assessing the success of any cancer treatment [14]. The gold standard endpoint, and the endpoint which is easiest to measure in a clinical trial, has always been overall survival. In patients with head and neck cancer, however, survival is not only impacted by the disease itself, but by the frequent underlining cardiopulmonary comorbidity, and by the significant incidence of second primary malignancy. In patients with advanced disease, an improvement in survival may be difficult to demonstrate, and may not be a prerequisite for symptomatic palliation. Tumor response, i.e., a measurable shrinkage in tumor volume, has always been considered to be an accurate reflection of antineoplastic activity [14]. Clear definitions of what actually constitutes a meaningful response are critically important in determining which chemotherapeutic agents might be of value in drug combinations, or in definitive multimodality treatment. These definitions have evolved over time but have been recently standardized as the Response Evaluation Criteria in Solid Tumors (RECIST) [15]. Although these criteria are important in allowing investigators to assess the efficacy of chemotherapy drugs and combinations, it should also be recognized that achievement of a formal response may not be necessary for a patient to achieve symptomatic benefit. There has been recent discussion about the value of “stable disease” as an endpoint of palliative systemic therapy [16]. Historically, if a chemotherapeutic drug was unable to produce actual tumor shrinkage, it was considered inactive, and the toxicity produced was not felt to be justified. With the recent proliferation of newer and better tolerated targeted therapies this has been called into question [17]. Many patients treated with these agents achieve disease stability without significant tumor shrinkage; and appear to benefit from continued treatment with a possible impact on survival. Thus the concept of “clinical benefit” (i.e., disease response and disease stability after treatment) has been legitimized as a meaningful endpoint in palliative management.

19  Principles of Systemic Chemotherapy for Squamous Cell Head and Neck Cancer

For patients being treated with curative intent, additional, more sophisticated endpoints are often chosen, including progression-free survival, disease-free survival, event-free survival, or disease-specific survival [18, 19]. Although these functions may be more reflective of the effect of treatment than the overall survival, they are often variably defined and difficult to interpret. Standard definitions have been proposed. When reporting the efficacy of local or regional treatment modalities, investigators have often chosen such endpoints as local or locoregional control [19]. While somewhat reflective of overall outcome, such assessments ignore the relationship between local, regional and distant disease, and do not fully address the overall impact of the disease on the patient. When measuring the effect of a systemic treatment, distant disease control is also a common endpoint. Once again, however, this function is not independent of locoregional control. Furthermore, distant metastases are a relatively infrequent cause of treatment failure in head and neck cancer. Even these endpoints may not be the most important outcome from the patient’s perspective. Cancers in the head and neck and their treatments may significantly compromise several major human functions including speech, swallowing, and nonstomal breathing. Preservation of these functions may be more important to a patient than survival. While organ preservation, i.e., the avoidance of surgical resection of the organ, is easy to measure, it is only a crude estimate of functional preservation, a more difficult endpoint to assess, particularly for any given patient [20]. Moreover, the acceptability of functional compromise will vary between patients, and functional restoration is often possible even after organ removal. Nonlaryngeal speech with preservation of swallowing, may or may not be a preferable outcome to speech preservation with feeding tube dependence for any given patient. List and colleagues from the University of Chicago have explored these kinds of patient-defined goals after head and neck cancer treatment in some detail [21]. When patients were asked to rank the relative importance of several treatment outcomes, cure and longer survival were consistently most important. There was considerable variability in the relative importance of other functional and cosmetic treatment priorities, including those goals related to pain, energy, voice, swallowing, and appearance. This is a message that we, as physicians, must remember when discussing treatments with our patients. A number of validated quality of life instruments have also been developed in an attempt to better assess the impact of treatment and disease from the patient’s perspective. Several of these tools have been widely employed including the Performance Status Scale for Head and Neck Cancer [22], the Functional Assessment of Cancer Therapy (FACT) scale [23], the EORTC quality of life questionnaire [24], and

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the University of Washington scale [25]. Thus far, however, the results and importance of these measurements are not entirely clear. When using chemotherapy as palliative treatment in patients with incurable disease, the acceptability of the acute toxicities is the major determinant of the risk/benefit ratio of the treatment. However, when chemotherapy is being used as part of a curative multimodality treatment approach, the acute toxicities, while important, are of less concern than any late or long-term morbidity. Fortunately, except for a small risk of sterility or of a second malignancy, late morbidity from chemotherapy is uncommon. It is clear, however, that the combination of chemotherapy and radiation increases the likelihood and severity of the long-term morbidities commonly associated with radiation, an interaction which must also be considered when choosing treatments [26].

General Considerations in the Use of Chemotherapy Most drugs used for systemic therapy in malignant disease exploit cancer cells' innate inability to repair genetic damage. Because normal cells in various tissues are vulnerable to these drug effects, chemotherapeutic agents are a class with a narrow therapeutic window. Preclinical models have demonstrated the steep dose–response curves after the administration of chemotherapy [27]. With any dose reduction of therapy, there is a consequent significant decrement in the degree of cancer cell kill and a resultant compromise in the ability to eliminate the malignant clone. The challenge in the delivery of chemotherapy is remaining within the therapeutic window, that is, being able to administer maximal drug doses while avoiding lethal injury to normal tissues. Chemotherapy is usually administered intermittently, but at regular time intervals so as to allow normal tissue (usually bone marrow) recovery from drug-related toxicity, and enable administration of adequate drug dose over time. As many chemotherapeutic agents are cell cycle specific, at any given time, a certain proportion of cancer cells are not in the chemotherapy-sensitive phase of the cell cycle. Apart from limiting toxicity, repeated drug exposure over time allows for surviving cancer cells to enter the specific cell cycle phase during which an agent exerts its antitumor effects. Due to consequences of the lifestyle that predisposes to head and neck cancer, cardiac, pulmonary and renal comorbidity, in addition to suboptimal compliance, complicate treatment planning in this subset of patients. Tailoring the choice of drug and treatment modality to patient factors is critical to optimizing treatment outcomes. The considerable acute toxicity of chemotherapy can result in significant

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­ orbidity and even mortality in patients who are poor m ­candidates for aggressive therapy. Pharmacokinetic considerations for this patient population also have to be taken into account when selecting the appropriate chemotherapeutic regimen. The oral route is often compromised in patients with advanced tumors of the head and neck, and the delivery and absorption of active orally administered drugs such as hydroxyurea may be impaired. Most chemotherapeutic drugs active in this disease are metabolized in the liver, and excreted through the biliary or renal route. Renal dysfunction, hepatic impairment, preexisting cardiovascular disease, and the frequency of considerable alcohol exposure, are all important considerations in the choice of chemotherapy. It is well recognized that previously untreated malignancies are more responsive to therapy than is persistent or recurrent local, regional, or distant disease after initial therapy. Certain molecular characteristics have been reported to predict for relapse after chemotherapy and radiation [28–30]. In addition to intrinsic variations in gene expression, persistent or recurrent head and neck cancers often acquire molecular aberrations from prior exposure to pharmacologic agents that render them more resistant to chemotherapy compared to treatment-naïve tumors [31, 32]. Changes in tumor vasculature from previous surgery or radiation, and increased expression of genes that promote hypoxic tumor growth are thought to contribute to radiation insensitivity [33]. These, in addition to the significant symptom burden of recurrent disease and prior therapy, magnify the difficulty of administering effective systemic therapy in this compromised patient population.

Single Agents: Mechanisms of Action, Toxicities, Metabolism The most frequently used agents in the treatment of both locally advanced and metastatic squamous cell head and neck cancer have been the platinum compounds, methotrexate,

5-fluorouracil, and the taxanes. All four drug classes have single agent activity, have differing mechanisms of action and toxicity, and can be administered concurrent with radiation as radiation sensitizers. Although many other antineoplastic drugs have known activity, the following section will focus on these four classes (Table 19.1). Cisplatin was the first platinum compound noted to have antitumor activity in head and neck cancer [34]. The mechanism of action is believed to be drug incorporation into DNA, forming DNA adducts which distort the normal DNA helical structure. This triggers cellular recognition of DNA damage and subsequent apoptosis. Increased intracellular cisplatin doses are noted when the drug is given with radiation. The systemic toxicity of cisplatin can be significant and involves multiple organ systems. It is highly emetogenic compound, which can cause both early and delayed chemotherapy-induced nausea and vomiting, now more easily controlled with modern effective antiemetic regimens. Nephrotoxicity through glomerular and renal tubular damage with resultant salt wasting can be a consequence of treatment. This can often be prevented and ameliorated by aggressive hydration. Peripheral neuropathy and irreversible ototoxicity (in the form of high frequency hearing loss) can also result from cumulative drug exposure. Carboplatin is an analogue of cisplatin, whose properties render it less nephro- and neurotoxic, but more myelotoxic than cisplatin. The chemical structure of carboplatin results in delayed drug conversion and excretion, resulting in a longer half-life than cisplatin. Both of these drugs are excreted primarily through the kidney [35]. The antifolates, like methotrexate, exert antitumor effects by impairing the cancer cell’s ability to generate precursors for DNA synthesis [36]. Methotrexate was approved for head and squamous cell cancer treatment in 1953. This drug inhibits dihydrofolate reductase, which maintains the intracellular supply of reduced folate essential for purine synthesis. Methotrexate has a wide range of systemic side effects, the most commonly observed are myelosuppression and gastrointestinal toxicity. Interstitial pneumonitis, hepatic transaminase elevation, and renal dysfunction from drug precipitation

Table 19.1  Commonly used chemotherapeutic agents in the management of head and neck cancer Class Agents Mechanism of action Clearance Platinum agents

Cisplatin Carboplatin

DNA adduct formation

Renal

Antifolates

Methotrexate

Renal

Antimetabolites

5-Fluorouracil

Taxanes

Paclitaxel Docetaxel

Depletes precursors for purine synthesis Depletion of precursors for DNA synthesis Incorporation into RNA Mitotic arrest by microtubule stabilization

Renal (inactive drug)

Hepatobiliary

Toxicity Nausea Nephro- and neurotoxicity Myelosuppression Myelosuppression Gastrointestinal toxicity Gastrointestinal toxicity Myelosuppression Hypersensitivity Myelosuppression Peripheral neuropathy

19  Principles of Systemic Chemotherapy for Squamous Cell Head and Neck Cancer

in the renal tubules are also recognized side effects. The majority of this drug is eliminated through the kidneys, with a small proportion, about 10%, excreted through the bile. 5-Fluorouracil is a uracil analog that impairs both DNA and RNA synthesis [37]. It is intracellularly converted to its active form, 5FdUMP, which inhibits the enzyme thymidylate synthetase, depleting thymidylate and arresting DNA synthesis. The drug can also be intracellularly converted into 5FUTP which, when incorporated into RNA, results in cell death. The drug has a short half-life lasting minutes, and can be administered as a bolus or infusion. Like methotrexate, 5-fluorouracil results in myelosuppression and gastrointestinal toxicity. Nausea, stomatitis, mucositis, and diarrhea are common manifestations. Coronary vasospasm resulting in myocardial infarction is a rare but reported side effect. This drug is degraded by the enzyme dihydropyrimidine dehydrogenase, which is present in most tissues. The inactive metabolites are excreted in the urine [38]. The taxanes, paclitaxel and docetaxel, are pharmacologic class of agents that induce cell death by stabilizing microtubule formation [39]. Subsequent metaphase arrest results in apoptosis. Both paclitaxel and docetaxel are primarily metabolized by the liver and excreted in the bile, thus appropriate dosage adjustments may be necessary in the setting of hepatic dysfunction [40]. Hypersensitivity reactions to paclitaxel are the most common acute toxicity, myalgias and arthralgias after drug administration are also common. Peripheral neuropathy is a cumulative side effect of both drugs. Docetaxel can result in fluid retention or skin toxicity.

Combination Chemotherapy: Rationale and Principles When single agents prove active in the management of a malignancy, the next step has always been an attempt to use these drugs in combination. The use of combination chemotherapy, however, is based on several clear principles [41] (Table 19.2). The first is that for a drug to be useful in a combination chemotherapy regimen, it must have single agent antineoplastic activity. It makes little sense to include an ineffective chemotherapeutic agent in a drug combination, with the hope that it will suddenly prove to kill cancer cells. It should be noted, however, that recent experience using some of the Table 19.2  Principles of combination chemotherapy 1. Drugs used in combination should have single agent activity 2. Drugs used in combination should be used in full therapeutic doses 3. Drugs used in combination should have nonoverlapping ­mechanisms of action 4. Drugs used in combination should have nonoverlapping toxicities

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targeted agents, most notably bevacizumab, has suggested that this caveat does not always hold true. Bevacizumab, a vascular endothelial growth factor (VEGF) inhibitor is a relatively ineffective antineoplastic agent when used alone. When used in combination with other chemotherapeutic drugs, however, it has a demonstrated benefit in several disease sites [42, 43]. The second general principle in the use of combination chemotherapy is the importance of using drugs in full therapeutic doses. There has been general recognition of a dose–response curve for most systemic chemotherapeutic agents. Larger doses tend to produce larger, if not exponentially larger, responses, and suboptimal dosing of multiple agents would be unlikely to produce a better result than the full therapeutic dose of a single drug. Third, drugs used in combination should have nonoverlapping mechanisms of action. There are a number of defined classes of chemotherapeutic agents, often with several different, but similar members. Rarely has the use of two drugs from the same class (e.g., two alkylating agents or two vinca alkaloids) been of any benefit. Finally, drugs, when used in combination should not have overlapping toxicities. In view of the steep dose–response curve for most chemotherapeutic agents, the optimal dosing for each drug is usually defined by its dose-limiting toxicity. Two drugs, with the same doselimiting toxicity (e.g., myelosuppression), if used at their maximally tolerated dose, will undoubtedly produce significant and perhaps intolerable toxicity and would be a poor combination. Despite the soundness of the rationale for combining chemotherapeutic agents, many of the common drug combinations used in this disease and others, violate one or several of these principles. Thus careful phase I and II testing for both toxicity and efficacy is important before widespread adoption of any chemotherapy combination.

Systemic Chemotherapy in Palliative Management Patients with persistent or recurrent disease not amenable to local therapy such as radiation or salvage surgery, or patients who develop or present with systemic metastasis are incurable. The prognosis for patients in this situation is dismal and there is little evidence suggesting that chemotherapy is superior to best supportive care. Survival in this patient group, even when palliative chemotherapy is administered, uniformly ranges from 6 to 9 months. In this situation, when cure and survival prolongation are not possible, the treatment goal is to palliate symptoms and improve quality of life. Quality of life can be adversely impacted by the local effects of tumors at both the primary site and the sites of metastasis. Local effects of the primary site tumor include

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pain, and impairment or loss of important functions such speech, swallowing, smell, hearing, and even vision. Cosmetic deformity in addition to functional compromise can cause significant body image issues and depression. Distant disease most often involves the lung, and less commonly bone. This can result in cough, hemoptysis, painful bone lesions, pathologic fractures, and nerve or spinal cord impingement. Palliative care to address these symptoms should be carried out by a multidisciplinary team. Modalities such as radiation therapy to painful sites, and adequate pain control contribute to palliation in the metastatic setting. Systemic chemotherapy is a widely used tool for reducing tumor burden, with the assumption that this leads to alleviation of tumor-related symptoms [44]. Active chemotherapy drugs when given as single agents often result in modest response rates ranging from 10 to 30% depending on previous treatment [45–47]. Several well-designed clinical trials have been done to compare various single and multiple drug regimens [48–50]. Although multiagent chemotherapy does produce a consistent increase in response rates, with only one exception, no significant prolongation of median survival has been observed. One of the more important observations has been the reproducible increase in treatment-related toxicity that accompanies combination drug therapy. This observation introduces a significant conflict with the palliative goals of care in a patient population with incurable disease and significant comorbidity. Certainly, the toxicity of chemotherapy would only be acceptable if it ultimately resulted in some alleviation of tumor-related symptoms. With little convincing evidence of a survival advantage with chemotherapy combinations, great care must be taken to appropriately select patients who are good candidates for combination treatment. In a patient with a compromised performance status, for example, combination chemotherapy may adversely impact quality of life rather than palliate symptoms. Phase III clinical trials using chemotherapy for patients with incurable disease carried out in the last two decades have focused on examining the endpoints of toxicity, survival and response rates. Little has been done to incorporate validated measurements of quality of life in these studies. The recognition that response rates may not accurately translate to improved symptom control, along with the introduction of a new class of “targeted agents” believed to have a more tolerable side effect profile, have led to the integration of more accurate quality of life measurements in the design of clinical trials. In general, among most solid tumors, the integration of new pharmacologic agents into curative intent therapy is initiated by observed drug activity in patients with recurrent, pretreated or metastatic disease. Some examples of these emerging drugs showing antitumor effects in the metastatic setting are newer generation nucleoside analogs, antifolates,

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and topoisomerase inhibitors. Gemcitabine is a novel ­synthetic pyrimidine analog which is activated through intracellular phosphorylation. In its activated form, it is incorporated into DNA and RNA and arrests their synthesis, it also inhibits its own inactivating enzyme, increasing intracellular concentrations [51]. The new generation antifolate pemetrexed inhibits several enzymes involved in the maintenance of reduced folate pools essential for the production of DNA precursors. Its property of rapid entry into the cellular environment through several transport mechanisms is known to overcome cellular resistance that often hampers the efficacy of older generation antifolates [52]. Irinotecan is a partly synthetic camptothecin, which inhibits topoisomerase I, causing supercoiling of DNA during replication and growth arrest [53]. These drugs have been shown to possess radation-sensitizing properties and their assimilation into curative treatment strategies awaits further investigation. The epidermal growth factor receptor and its demonstrated synergistic activity with both chemotherapy and radiation resulted in studies using the EGFr inhibitors in the metastatic setting. When compared to single agent methotrexate, EGFr inhibitors used alone have had disappointing response rates and no demonstrable impact on survival [54]. However, recently published data on the combination of platinum-based chemotherapy and EGFr inhibition has shown an unprecedented albeit modest improvement in survival [55]. The combination of chemotherapy with targeted agents has demonstrated a similar survival advantage in other epithelial malignancies and may represent the future paradigm for investigating and treating metastatic disease.

Systemic Chemotherapy in Definitive Management In the curative management of solid tumors, single modality chemotherapy is rarely sufficient. For most neoplasms, and in particular head and neck cancers, chemotherapy is only effective when used in combination with definitive radiation therapy and/or surgery. Chemotherapy must be considered adjunctive not curative, and its use in multimodality treatment regimens must not compromise the delivery of the definitive locoregional treatment. While considerable morbidity may be acceptable from aggressive curative treatment regimens, the toxicity produced by the addition of chemotherapy cannot be allowed to interfere with the required radiation or surgery. A number of multimodality treatment approaches have been explored (Table 19.3). All have been based on the ­recognized chemosensitivity of head and neck cancer. Previously untreated patients with squamous cell head and  neck cancer can be expected to respond to systemic

19  Principles of Systemic Chemotherapy for Squamous Cell Head and Neck Cancer Table 19.3  Multimodality treatment approaches using chemotherapy Induction chemotherapy The use of chemotherapy prior to definitive locoregional management Adjuvant chemotherapy The use of chemotherapy after definitive locoregional management Concurrent chemoradiotherapy Definitive The use of concomitant chemotherapy chemoradiotherapy and radiation as definitive management Adjuvant The use of concomitant chemotherapy chemoradiotherapy and radiation after definitive locoregional management Sequential treatment The use of induction chemotherapy followed by definitive concomitant chemotherapy and radiation

c­ ombination chemotherapy up to 90% of the time, with ­complete responses described in between 30 and 50% of patients. These excellent responses are rarely durable, however, and disease regrowth is the rule. The question then becomes how best to exploit this antineoplastic activity in conjunction with definitive radiation and surgery. Induction chemotherapy was the first treatment strategy developed. The rationale for induction chemotherapy was that given the increased chemotherapy responsiveness in the previously untreated patient, the optimal time to use chemotherapy would be prior to any locoregional intervention. It was reasoned that if significant tumor shrinkage could be achieved, there might, as well, be an improvement in locoregional control, a decrease in distant metastasis, and an overall survival improvement. The potential for surgical modification or organ preservation after chemotherapyinduced tumor shrinkage was also suggested. An alternative strategy is the use of adjuvant, or postoperative chemotherapy. Adjuvant chemotherapy strategies are meant to address concern about disease recurrence, and are optimal for those patients likely to develop distant metastasis even after achieving locoregional control. Thus a patient identified as being at high risk for distant disease recurrence after definitive surgery and or radiation might be appropriate for further systemic chemotherapy. Not surprisingly, given the limited risk for distant metastases in this disease, single modality adjuvant chemotherapy has not been of major benefit. Several observations emerged from these kinds of sequential treatment approaches, however. The first was the recognition that chemotherapy responsiveness was predictive for responsiveness to radiation therapy [56]. This suggested the potential that chemotherapy might serve as a selection tool to identify those patients most likely to benefit from radiotherapeutic (i.e., nonoperative) intervention [57]. Chemotherapy was also found to decrease the risk of distant metastases, an achievement with a limited survival impact in a disease with such a small risk for distant disease [58–60]. Unfortunately, it was also recognized that treatment compliance could be

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compromised by successful induction chemotherapy. The dramatic response to systemic chemotherapy often experienced by these patients on occasion led to a motivational interference with completion of definitive treatment. The observation was also made that those patients who respond to systemic chemotherapy live longer than those patients who do not. This has been suggested by some as a justification for the use of systemic chemotherapy. It must be recognized however, that a response to chemotherapy is more common in those patients with a better performance status and smaller disease burden. These are also the patients with a better prognosis irrespective of the treatment utilized [61]. An alternative to the sequential use of single treatment modalities has been the concurrent use of chemotherapy and radiation. The rationale for this approach has been the recognition that both chemotherapy and radiation therapy are independently active treatment modalities and that chemotherapy may potentiate radiation, improve locoregional control, and decrease the impact of distant micrometastatic disease. In addition, the use of these two treatment modalities together, rather than sequentially, will shorten the overall treatment duration and in theory improve compliance. Preclinical data support a synergistic role of chemotherapy and radiation therapy through various postulated mechanisms. The enhanced cell kill from simultaneous exposure to systemic chemotherapy and radiation has been attributed to increased cellular cytotoxic drug uptake during radiation, chemotherapy-induced impairment of DNA repair mechanisms in response to radiation-induced damage, and ­chemotherapy-induced cell cycle shift resulting in increased radiation sensitivity. There are also several disadvantages to the concomitant use of chemotherapy and radiation. Clearly, the concurrent use of two treatment modalities will produce greater toxicity than the use of either treatment modality alone. This toxicity may then result in a compromise of dose intensity and efficacy, such as single agent rather than combination chemotherapy, split rather than continuous course radiation, or a reduction of the chemotherapy doses used. Nonetheless, the concurrent use of chemotherapy and radiation has been intensively explored in this disease both as definitive management, and as a postoperative adjuvant. Both locoregional control and survival have been improved with this approach although the treatment has been associated with significant acute and late toxicity [62]. Along with this improvement in locoregional control has been the recognition of a relative increase in the frequency of distant metastases, a change in the natural history of this ­disease [63, 64]. Given the apparent benefit achieved by induction chemotherapy in reducing the risk of distant metastasis, it has been recently suggested that a sequential treatment approach of induction chemotherapy followed by concurrent chemoradiotherapy might be advantageous [65].

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The induction chemotherapy would address the risk of ­distant metastasis and the concurrent chemoradiotherapy would deal with the locoregional disease. Randomized studies of this treatment schedule are currently underway. Critical to the use of systemic chemotherapy, both with and without radiation, has been the integration with surgery. Optimal management of the primary site and of the neck requires the definition of careful treatment algorithms. Patients with persistent or recurrent primary site disease after chemoradiotherapy will require some kind of surgical salvage. Patients presenting with large neck nodes at diagnosis, or with neck nodes that only incompletely respond to nonoperative intervention, will require subsequent neck dissection with curative intent [66]. Given the potential for cure after such surgical salvage, it would seem important that we be able to identify those patients likely to fail in the neck or at the primary site after nonoperative intervention. The development of organ preservation strategies has been somewhat unique to this field. The rationale for organ preservation is the hope that the substitution of radiation, with or without chemotherapy, for surgery might not compromise survival and yet preserve organ integrity and function. The goal of treatment is no longer one of an improved survival. Instead, it is the hope that survival will not be compromised, but that there will be more organ (usually larynx) preservation. Again it is important to point out the difference between organ preservation and organ function preservation [20]. Preservation of a nonfunctional larynx is of little benefit to a patient despite maintenance of its anatomic integrity. Studies of both induction and concurrent chemotherapy and radiation schedules have been conducted with some success. However, recent data has raised the possibility that current organ preservation practices may have compromised overall survival in larynx cancer [67]. Thus, for any given patient, the debate about the relative importance of organ preservation vs. survival continues.

Emerging Issues Increasing understanding of the molecular processes underlying head and neck squamous cell cancers, the discovery of new therapeutic targets, and the changing disease epidemiology has had a great impact on current scientific inquiry into the role of chemotherapy in improving patient outcomes. The decreasing popularity of tobacco use has resulted in a plateau and decline of most tobacco-related malignancies of the upper aerodigestive tract [68]. Among head and neck cancers, a distinct clinical entity of high-risk HPV-positive oropharyngeal head and neck cancers in a patient population without exposure to tobacco or alcohol has surfaced. These tumors have a different molecular profile and have improved

C.P. Rodriguez and D.J. Adelstein

prognosis compared to non-HPV-related squamous cell malignancies of the head and neck [69]. These patients are younger with less comorbid conditions, and respond to definitive therapy with excellent local and distant control rates. The applicability of previously established therapies for head and neck cancer to this previously unrecognized clinical entity has been called into question, and a reduction of the intensity of therapy to spare patients from the attendant toxicity of chemotherapy and radiation combinations has been proposed for this patient population. Contemporary clinical studies are now moving toward studying HPVpositive and negative head and neck cancers separately, to further define the appropriate therapy for these two distinct subsets of patients. Since the discovery that inhibiting the bcr-abl tyrosine kinase results in dramatic responses in patients with CML, numerous molecular markers have been identified as therapeutic targets in head and neck cancer. Inhibiting the epidermal growth factor receptor has been shown to result in synergistic cell kill when used with radiation and chemotherapy [70]. The combination of the monoclonal antibody cetuximab with definitive radiation in locally advanced head and neck squamous cell carcinomas have been shown to be superior to radiation alone in a large phase III clinical trial, with no significant increase in treatment-related toxicity [71]. Another phase III trial comparing combination chemotherapy to the same chemotherapy with cetuximab in patients with recurrent metastatic head and neck cancer demonstrated a modest survival advantage; an observation never before made in clinical trials using chemotherapy combinations alone [55]. The generally more favorable toxicity profile of these agents make them attractive prospects for integration into definitive and palliative therapy, and they are currently under study. Another emerging role for systemic therapy is in salvage treatment for recurrent or persistent disease. Historically, when a patient experiences locoregional failure after definitive chemotherapy and radiation, surgery, when possible, was the only potentially curative option for salvage therapy. With the advent of more sophisticated radiation therapy techniques, reirradiation has been shown to be a feasible and successful in a highly select group of patients. Because of the dose and field limitations imposed by prior radiation therapy, reirradiation with the addition of systemic therapy for radiation sensitization is an attractive prospect. Several phase II studies have demonstrated the tolerability and efficacy of this approach [72, 73]. Sensitivity to chemotherapy is generally thought to identify disease with a more favorable disease biology. Complete responses to systemic therapy in most solid tumor malignancies are almost always associated with improved outcomes. Because the acute and long-term toxicities of surgery and chemoradiation are substantial, the possibility of using chemotherapy alone

19  Principles of Systemic Chemotherapy for Squamous Cell Head and Neck Cancer

to select and cure local disease is being investigated. Single institution clinical ­studies have explored the use of chemotherapy alone for nonmetastatic laryngeal carcinoma and demonstrated long-term disease remission in a subset of patients [74, 75]. Results of further studies are awaited before this strategy becomes applicable to clinical practice.

Conclusion The current role of chemotherapy in the definitive management of head and neck cancer has been established by extensive scientific investigation over many decades. The benefits and toxicities of these agents have been well defined. The identification of molecular therapeutic targets, the development of novel active agents, and the changing epidemiology and treatment failure patterns of head and neck cancer are providing avenues for expanding the application of systemic therapy to improve outcomes in both local and metastatic disease.

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19  Principles of Systemic Chemotherapy for Squamous Cell Head and Neck Cancer 69. Fakhry C, Westra WH, Li S, et  al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst. 2008;100:261–9. 70. Holsinger FC, Doan DD, Jasser SA, et al. Epidermal growth factor receptor blockade potentiates apoptosis mediated by paclitaxel and leads to prolonged survival in a murine model of oral cancer. Clin Cancer Res. 2003;9:3183–9. 71. Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 2006;354:567–78. 72. Langer CJ, Harris J, Horwitz EM, et al. Phase II study of lowdose paclitaxel and cisplatin in combination with split-course concomitant twice-daily reirradiation in recurrent squamous

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Chapter 20

Molecular Targeted Therapies in Head and Neck Cancer Vinai Gondi, Anne M. Traynor, and Paul M. Harari

Abstract  Molecular targeted therapies for head and neck cancer offer promising opportunities to improve upon clinical outcomes of conventional treatments. The toxicity profiles of these agents are unique, reflecting their distinct mechanisms of action, and generally do not appear to directly overlap or amplify the toxicities of conventional treatment modalities. Herein, we discuss strategies for targeting the epidermal growth factor receptor (EGFR) and angiogenesis (VEGF) as models for the development and clinical evaluation of molecular targeted agents. We explore phase III clinical data demonstrating measurable survival benefits with the use of cetuximab in the locally advanced and recurrent/metastatic head and neck cancer settings. In addition, we review EGFR tyrosine kinase inhibitors and angiogenesis inhibitors, both of which have shown activity in head and neck cancer. Finally, we review promising new agents and future directions for the incorporation of molecular targeted therapies in head and neck cancer. Keywords  Targeted therapies • EGFR • Angiogenesis • Head and neck cancer • Bevacizumab • Cetuximab • Tyrosine kinase inhibitors • Erlotinib • Gefitinib

Introduction Conventional treatment of head and neck squamous cell carcinoma (HNSCC) frequently involves a combination of surgery, radiotherapy, and/or chemotherapy. However, the degree to which these treatments can be successfully implemented is commonly limited by toxicity. Targeted therapies, due to their specificity for molecular tumor targets, offer the potential to improve upon outcomes of conventional treatments without significantly amplifying treatment-limiting toxicity profiles.

Herein, we discuss strategies for targeting the epidermal growth factor receptor (EGFR) and angiogenesis (VEGFR) as models for the development and clinical evaluation of molecular targeted agents and briefly explore future directions of targeted therapies in head and neck cancer.

Targeting the Epidermal Growth Factor Receptor The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases and has been recognized as an important therapeutic target for a broad spectrum of epithelial tumors [1, 2]. The importance of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinoma is supported by its overexpression in the vast majority of head and neck tumors [3–12] and the predictive and prognostic value of EGFR expression level [13–16]. In the early 1980s, EGFR signaling blockade was postulated as a potential anticancer treatment strategy [17], and a series of EGFR inhibitors were subsequently developed with translational and clinical testing of lead agents in recent years [18, 19]. Cetuximab, a chimeric human:murine monoclonal antibody that prevents the binding of endogenous EGFR ligand to the extracellular domain of EGFR, represents one such class of EGFR inhibitor. The other primary class of EGFR inhibitor, tyrosine kinase inhibitors such as erlotinib and gefitinib, reversibly inhibit the catalytic activity of EGFR by binding the adenosine triphosphate (ATP) binding site of the EGFR kinase domain (Fig. 20.1).

Cetuximab Preclinical Data

P.M. Harari (*) Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison, WI, USA e-mail: [email protected]

Preclinical HNSCC models have demonstrated important cellular effects of cetuximab, including downregulation and internalization of EGFR, enhanced apoptosis, and inhibition

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_20, © Springer Science+Business Media, LLC 2011

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Fig. 20.1  EGFR targeting with monoclonal antibodies (mAb) or tyrosine kinase inhibitors (TKI) and subsequent cellular and tissue effects. From Harari, Huang. Clinical Cancer Research, Online. Copyright 2004 by American Association for Cancer Research. Reproduced with permission of American Association for Cancer Research

of cell cycle proliferation, cell migration, and tumor angiogenesis [18, 19]. Furthermore, cetuximab has demonstrated important chemo- and radio-sensitization effects [20–23], highlighting the potential value of EGFR signal modulation in tumor response to conventional cancer treatments. The chemo- and radio-sensitizing properties of cetuximab have been clinically validated in recent phase III clinical trials in both the definitive and metastatic/recurrent settings.

Locoregionally Advanced HNSCC In the curative treatment setting, a recent phase III international trial compared radiotherapy plus cetuximab to radiotherapy alone in patients with stage III–IV nonmetastatic HNSCC of the oropharynx, hypopharynx, or larynx [24]. Other eligibility criteria included medical suitability for definitive radiotherapy, Karnofsky performance score ³60, and normal hematopoietic, hepatic, and renal function. A total of 424 eligible patients were enrolled and randomized to either radiotherapy alone or concurrently with cetuximab. Given the global variance in preference for radiation fractionation at the time of trial design, investigators were permitted to employ conventional fractionation, hyperfractionation, or concomitant boost radiotherapy schedules. Cetuximab was initiated 1 week before radiotherapy at a loading dose of

400  mg/m2, followed by weekly infusions of 250  mg/m2. Patients were stratified according to Karnofksy performance status, nodal involvement, tumor stage, and radiation fractionation regimen. The primary endpoint was locoregional control, with secondary points including overall survival, progression-free survival, overall response rate, and toxicities. After median follow-up of 54 months, the addition of cetuximab improved locoregional control (median duration of locoregional control 24.4 vs. 14.9 months, p < 0.01), progression-free survival (p = 0.006) and overall survival (median survival 49.0 months vs. 29.3 months, p = 0.03) (Fig.  20.2). Importantly, this therapeutic benefit was achieved without exacerbation of radiotherapy-related toxicities, such as mucositis, xerostomia, pain, weight loss, or performance status deterioration (Table  20.1a). The addition of cetuximab did increase the rate of Grade ³3 acneiform rash or infusion reaction, both cetuximab-related toxicities, but notably did not adversely alter quality of life [25]. Long-term data from this trial with 5-year median follow-up confirms stability of the locoregional control and overall survival benefit with the addition of cetuximab to radiotherapy [26]. This study was the first to provide proof of principle that a molecular targeted agent could improve survival outcome without amplifying treatment-limiting toxicities in HNSCC. Interestingly, the benefit of cetuximab combined with radiotherapy manifested most strongly with the use of

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295 Table  20.1  Comparison of grade ³3 toxicities in (a) phase III international trial of radiotherapy (RT) with or without cetuximab for patients with stage III–IV HNSCC, and (b) the EXTREME phase III trial of platinum-based doublet chemotherapy with or without cetuximab as first-line treatment for recurrent or metastatic HNSCC (a) RT alone RT + Cetuximab Adverse event (N = 212) (N = 208) P value Percent of patients Mucositis 52 56 0.44 Xerostomia 3 5 0.32 Dysphagia 30 26 0.45 RT dermatitis 18 23 0.27 Weight loss 7 11 0.12 Asthenia 5 4 0.64 Acneiform rash 1 17 <0.001 Infusion reaction 0 3 0.01 From Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 2006;354:567–578. Copyright © 2006 Massachusetts Medical Society. All rights reserved (b) Chemo Chemo + Cetuximab Adverse event (N = 215) (N = 219) P value Percent of patients Neutropenia 23 22 0.91 Anemia 19 13 0.12 Thrombocytopenia 11 11 1.00 Leukopenia 9 9 1.00 Skin reaction <1 9 <0.001 Sepsis <1 4 0.02 From Vermorken JB, Mesia R, Rivera F, et al. Platinum-based chemotherapy plus cetuximab in head and neck cancer. N Engl J Med. 2008;359:1116–1127. Copyright © 2008 Massachusetts Medical Society. All rights reserved

Fig.  20.2  Kaplan–Meier estimates of locoregional control (a) and overall survival (b) in the phase III international trial of radiotherapy with or without cetuximab for patients with stage III–IV HNSCC. The dotted lines indicate median duration of locoregional control (A; 24.4 months vs. 14.9 months, p < 0.01) and median survival (B; 49.0 months vs. 29.3 months, p = 0.03). From Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 2006;354:567–578. Copyright © 2006 Massachusetts Medical Society. All rights reserved

c­ oncomitant boost radiotherapy (n = 120, HR = 0.62) or hyperfractionation technique (n = 37, HR = 0.74), but not with conventional fractionation (n = 55, HR = 1.01). There are data to suggest that HNSCC tumors with high EGFR expression may derive greater benefit with accelerated radiotherapy [27], pointing toward a relationship between EGFR signaling and altered radiation fractionation. However, caution is advised in interpreting clinical observations that derive from subset analyses. Further advances in our understanding

of the survival benefits of altered radiation fractionation [28–30] and concurrent chemoradiotherapy [28, 31–33], contributed to the development of phase II trials [34, 35] and a subsequent ongoing phase III trial (RTOG 0522) combining cetuximab with concurrent cisplatin and accelerated radiotherapy. In this trial, patients with stage III–IV nonmetastatic HNSCC of the oropharynx, hypopharynx, or larynx are randomized to cisplatin-based chemoradiotherapy with or without cetuximab. Radiation therapy is delivered in an accelerated fashion, with patients stratified according to whether three-dimensional conformal radiotherapy or intensity-modulated radiotherapy is utilized. The primary endpoint is progression-free survival, and secondary endpoints include overall survival, locoregional control, acute and late toxicities, and quality of life and health economic endpoints. Additional phase II trials combining cetuximab with chemoradiotherapy are also ongoing in the postoperative (RTOG 0234) and unresectable (ECOG 3303) settings.

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Metastatic/Recurrent HNSCC The role of cetuximab in platinum-refractory metastatic/ recurrent HNSCC has been investigated in three multicenter phase II trials. In one phase II trial, a total of 103 patients with platinum-refractory metastatic/recurrent HNSCC received cetuximab weekly until disease progression. The overall response rate was 13%, with all responders manifesting partial responses [36]. This response rate was similar to the response rate observed in other phase II trials of metastatic/recurrent HNSCC, in which cetuximab was added to the platinum-based chemotherapy regimen on which patients were failing [37, 38]. The similar response rates between cetuximab alone and cetuximab with chemotherapy in platinum-refractory HNSCC contrasted with prior observations in irinotecan-refractory metastatic colorectal cancer, in which cetuximab added to irinotecan showed improved activity compared to cetuximab alone [39]. In the first-line treatment of metastatic/recurrent HNSCC, an ECOG phase III study randomized 117 patients to cisplatin alone (100  mg/m2 every 4 weeks) or with cetuximab (400 mg/m2 cycle 1, followed by 250 mg/m2 weekly) [40]. After a median follow-up of 31 months, the addition of cetuximab to cisplatin significantly improved response rate (26% vs. 10%, p = 0.03), but did not significantly alter ­progression-free survival (primary study endpoint) or overall survival. The better than anticipated survival of patients in the cisplatin arm rendered this study underpowered to uncover a statistically significant improvement in progressionfree survival despite the strong improvement in tumor response with the addition of cetuximab. During the accrual of this trial, phase II studies involving cetuximab therapy in colon cancer [41] and other EGFR inhibitors in head and neck cancer [42], observed an intriguing correlation between the development of skin toxicity and biologic activity to EGFR inhibition. Such a correlation was also investigated in this ECOG study. Consistent with prior literature, the development of a cetuximab-related skin reaction was correlated with an improvement in overall survival (HR 0.42, p = 0.01). After phase II data demonstrated activity combining 5-fluorouracil with cetuximab and cisplatin [43], the EXTREME phase III trial was designed to investigate the efficacy of this regimen as first-line therapy for metastatic/ recurrent head and neck cancer [44]. Patients were included if their disease was considered unsuitable for local therapy and excluded if they received prior systemic chemotherapy less than 6 months, or surgery or radiotherapy less than 4 weeks, prior to study entry. A total of 442 patients underwent randomization at 81 centers in 17 European countries. Platinum-based chemotherapy involved a maximum of 6 cycles of either cisplatin (100 mg/m2 on day 1) or carboplatin (area under the curve of 5 mg/ml on day 1) plus 5-fluorouracil (1,000  mg/m2 per day for 4 days), delivered every

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3 weeks. Cetuximab was administered at a dose of 400 mg/ m2 initially, followed by weekly infusions of 250 mg/m2. In the cetuximab arm, patients with stable disease continued to receive cetuximab until disease progression or unacceptable toxicities, whichever occurred first. Patients in the chemotherapy-alone group received no further active treatment. Of the 413 tumors tested by immunohistochemical analysis, 98% had detectable EGFR, with 40% or more EGFR-positive cells observed in 80% of tested tumors. The addition of cetuximab to the platinum-based doublet regimen improved response rate (35.6% vs. 19.5%, p = 0.0001) and prolonged median progression-free survival (5.6 months vs. 3.3 months, p < 0.001) and median overall survival (10.1 months vs. 7.4 months, p = 0.04) (Fig. 20.3) without exacerbation of chemotherapy-related hematologic toxicities or quality of life (Table  20.1b). Preliminary analysis of EGFR gene copy number, assayed by fluorescent in-situ hybridization (FISH), was not predictive of cetuximab efficacy [45].

Toxicities A greater understanding of cetuximab-related toxicites has emerged in recent years, as its clinical use has become more widespread. Cetuximab-related toxicities generally involve skin and allergic reactions. Encompassing multiple different manifestations, the skin rash associated with cetuximab is most frequently acneiform in appearance and generally distributed in skin areas rich in sebaceous glands, such as the face, neck, shoulders, upper trunk, and scalp (Fig. 20.4). The development of the rash is largely attributed to high levels of EGFR expression in the epidermis and hair follicle. Approximately 70% cetuximab-related skin reactions are grade 1 or 2 [24] and resolve without the need for pharmacologic intervention. Both in head and neck cancer [40] and other tumor sites [46], the development of a cetuximabrelated rash appears to correlate with improved activity. As a chimeric mouse–human IgG1 monoclonal antibody, cetuximab is associated with allergic and occasional anaphylactic reactions. Though the product label indicates that severe hypersensitivity reactions occur in approximately 3% of patients, higher rates have been reported in a few distinct geographic regions including the southeast USA and in Sydney, Australia, as compared to other areas [39, 47–49]. Many of these reactions occur within minutes after a patient’s first exposure to the drug, compatible with IgE-mediated reaction. Recent data has observed an association between cetuximab-related reactions and the presence of IgE antibodies directed against galactose-a(alpha)-1,3-galactose prior to the first infusion with cetuximab (Fig. 20.5). Prior exposure to galactose-a(alpha)-1,3-galactose may induce the generation of galactose-a(alpha)-1,3-galactose-specific IgE antibodies

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Fig. 20.3  Kaplan–Meier estimates of overall survival (a) and progression-free survival (b) in the EXTREME phase III trial of platinum-based doublet chemotherapy with or without cetuximab in the first-line treatment of recurrent or metastatic HNSCC. The addition of cetuximab to the platinumbased doublet regimen prolonged median progression-free survival (5.6 months vs. 3.3 months, p < 0.001) and median overall survival (10.1 months vs. 7.4 months, p = 0.04) From Vermorken JB, Mesia R, Rivera F, et al. Platinum-based chemotherapy plus cetuximab in head and neck cancer. N Engl J Med. 2008;359:1116-1127. Copyright © 2008 Massachusetts Medical Society. All rights reserved

in some patients, placing these patients at increased risk for cetuximab-related anaphylactic reactions [50]. In contrast to cetuximab, two other anti-EGFR monoclonal antibodies, panitumumab and zalutumumab, are fully human and therefore carry less theoretical risk for allergic reactions. Preclinical studies have shown augmentation of radiation response using panitumumab in HNSCC [51]. Phase II/III trials using panitumumab in the locally advanced and ­metastatic/recurrent settings, and phase III trials using zalutumumab in the locally advanced and platinum-refractory settings, are ongoing [52].

EGFR Tyrosine Kinase Inhibitors Small-molecule tyrosine kinase inhibitors (TKIs) bind to the intracellular tyrosine kinase domain of EGFR and inhibit phosphorylation, thereby blocking downstream signal transduction (see Fig.  20.1). These agents have demonstrated antitumor activity in multiple preclinical models [53–61]. Of the anti-EGFR TKIs, gefitinib, and erlotinib are the furthest along in their clinical development and, unlike monoclonal antibodies, can be administered orally, most commonly on a once daily basis. The phase III Iressa vs. Methotrexate or

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Fig. 20.4  Cetuximab-related acneiform rash. A 48-year-old man with metastatic colon cancer was treated with fluorouracil, leucovorin, oxaliplatin, and cetuximab. An acneiform eruption developed on his face, chest, and back (Panel A). After gentle débridement, the right half of his face was treated with 0.2% hydrocortisone valerate and the left treated with 0.1% tazarotene. One week later, there was greater

Fig. 20.5  Structure of cetuximab. Galactose-a(alpha)1,3-galactose oligopolysaccharides are located on the murine Fab portion, and not the human Fc portion. IgE antibodies directed at this antigen on cetuximab may predispose patients to cetuximab-related anaphylactic reactions. S-S denotes a disulfide bond. Adapted from Chung CH, Mirakhur B, Chan E, et al. Cetuximab-induced anaphylaxis and IgE specific for ­galactose-alpha-1,3-galactose. N Engl J Med. 2008;358: 1109-1117. Copyright © 2008 Massachusetts Medical Society. All rights reserved

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improvement on the right side (Panel B), suggesting that anti-inflammatory agents may prove effective for the skin toxicity associated with this class of agent. Adapted from Moss JE and Burtness B. Cetuximab-associated Acneiform Eruption. N Engl J Med. 2005; 353 (19). Copyright © 2005 Massachusetts Medical Society. All rights reserved

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“IMEX” trial randomized 486 patients with recurrent/metastatic HNSCC to gefinitib 250 or 500 mg/day or methotrexate [62]. No benefit was observed in objective response rate (2.7%, 7.6%, 3.9%, respectively), quality of life (13.4%, 18.0%, 6.0%, respectively), or the primary endpoint of overall survival (median overall survival, 5.6, 6.0, 6.7 months, respectively). A phase III ECOG study was designed to randomize 330 patients with recurrent/metastatic HNSCC to docetaxel with or without gefitinib. The study was terminated early after an interim analysis demonstrated a low likelihood of reaching an overall survival benefit, the primary endpoint. Preliminary analysis of the 270 enrolled patients demonstrated prolongation of time to progression with the addition of gefitinib (median, 3.5 and 2.0 months with and without gefitinib, respectively), but no improvement in overall or progression-free survival [63].

Fig. 20.6  Angiogenic switch. Most tumors start as avascular nodules (dormant) (a) until equilibrium is reached between rate of proliferation and apoptosis. The “angiogenic switch,” once activated, leads to perivascular detachment and vessel dilation (b), angiogenic sprouting (c), new vessel formation and maturation (d), and ultimately mature tumor vasculature (e). Adapted from Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer. 2003;3:401–10. With permission from Nature Publishing Group

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Targeting Angiogenesis In 1971 Folkman described the ability of tumors to form new blood vessels, termed angiogenesis, and implicated the importance of this process for tumor growth and progression [64]. Initial preclinical data demonstrated the inability of solid tumors to grow larger than 2–3  mm or to generate metastases in the absence of new blood vessels [65–67]. Without angiogenesis, tumor cells reach equilibrium between the rate of proliferation and death [68]. To recruit their own blood supply, tumor cells must activate the “angiogenic switch,” a term coined to describe a shift in the peri-tumoral homeostasis between pro- and antiangiogenic factors (Fig.  20.6) [67, 69]. Preclinical data has demonstrated that this process occurs early in cancer development and ­represents a rate-limiting step in tumor progression [70].

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Triggers of the “angiogenesis switch” include hypoxia, which manifests as the tumor cell outgrows its existing blood supply. The net product of angiogenesis is the generation of new tumor vessels that are structurally abnormal, rendering the vessels “leaky” and inefficient in their ability to deliver blood, oxygen, and nutrients. This, in turn, results in further hypoxia, creating a feedback loop of perpetually activated angiogenesis [71]. One of the prime angiogenic targets, vascular endothelial growth factor (VEGF), is thought to play a central proangiogenic role in HNSCC [72–74]. VEGF binds to its transmembrane tyrosine kinase receptor VEGFR-2 to activate signal transduction, which ultimately stimulates vascular endothelial cell proliferation and survival and secretes proteolytic enzymes to break down extracellular matrix [67, 75]. The ubiquity of VEGF and VEGFR in HNSCC, expressed in 90% of HNSCC tumor samples [76, 77], renders the VEGF signaling pathway an attractive biologic target. In an analysis of HNSCC tumor samples from 1,002 patients in 12 studies, VEGF immunohistochemical staining was associated with a 1.88-fold higher risk of death at 2 years [78]. In addition, increased VEGF levels in HNSCC tumor samples and patient serum have been associated with tumor growth, metastasis, and treatment failure [79–82]. In preclinical HNSCC models, inhibition of the VEGF signaling pathway has markedly decreased angiogenesis, inhibited tumor growth, and augmented radiation response [83–85]. The latter effect is thought to be due, in part, to the ability of VEGF inhibition to prevent the development of structurally abnormal blood vessels. By normalizing tumor vasculature in this way, VEGF inhibition is thought to mitigate tumor hypoxia, a known marker of radioresistance [86].

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Combining Anti-VEGF and Anti-EGFR Therapies Based on preclinical data demonstrating a favorable interaction between anti-VEGF and anti-EGFR therapies [87] and the potential for anti-VEGF therapies to reverse EGFR resistance [88, 89], a phase I/II trial of combining bevacizumab with erlotinib in the recurrent/metastatic HNSCC setting was conducted [71]. In the phase I component, ten patients were enrolled in three successive cohorts with no dose-limiting toxicities. In the phase II component, 46  patients were enrolled on the highest dose of bevacizumab (15 mg/kg every 3 weeks). Median overall survival and progression-free survival were 7.1 and 4.1 months, respectively. The overall response rate was 14.6%, with 4  patients demonstrating a complete response. Common toxicities included rash and diarrhea from erlotinib. Three patients had severe bleeding events of grade 3 or higher. In the locally advanced setting, a phase II trial of induction chemotherapy of paclitaxel, carboplatin, 5-FU, and ­bevacizumab for two cycles, followed by concurrent chemoradiotherapy involving paclitaxel, bevacizumab, and erlotinib, enrolled 60 patients. Preliminary analysis demonstrated an 18-month progression-free survival and overall survival of 85 and 87%, respectively, after a median followup of 16 months [90]. In the locally advanced setting, the benefit of adding bevacizumab to chemoradiotherapy involving pemetrexed and cetuximab is being studied in a phase II trial, with progression-free survival as the primary endpoint [52].

Multikinase Inhibitors Bevacizumab Bevacizumab is a fully humanized monoclonal antibody specific to all isoforms of the VEGFA ligand. Approved by the FDA for use in metastatic colorectal cancer and non-small-cell lung cancer. Bevacizumab is the most mature agent in clinical testing of antiangiogenic therapies for HNSCC. In an ongoing phase III trial conducted by the Eastern Cooperative Oncology Group (ECOG), 400 patients with treatment-naïve recurrent/­metastatic HNSCC are being randomized to docetaxel/cisplatin/5-FU with or without bevacizumab with overall survival as the primary endpoint [52]. Several phase I trials are underway examining the feasibility of incorporating bevacizumab into concurrent chemoradiation regimens for curative HNSCC patients.

By blocking multiple biologic targets, multikinase inhibitors have the potential to inhibit multiple signaling pathways as a single oral agent. Sorafenib, sunitinib, and vandetanib are furthest in their clinical testing in HNSCC. Sorafenib inhibits the activity of VEGFR, platelet-derived growth factor receptor (PDGFR), and the RAF/MEK/ ERK signaling pathways, all of which are associated with tumor angiogenesis and tumor growth and proliferation. Sorafenib monotherapy in treatment-naïve recurrent/­metastatic HNSCC was associated with median overall survival and time to progression of 8 and 4 months, respectively [91]. Sunitinib, FDA-approved for renal cell carcinoma, also inhibits multiple kinases, including VEGFR, PDGFR, c-kit, FLT3, to simultaneously inhibit angiogenesis and tumor proliferation. Preliminary results from GORTEC 2006-01, a phase II study of sunitinib monotherapy in

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Table 20.2  Common toxicities associated with antiangiogenic agents Toxicity Frequency Possible underlying mechanism Hypertension 15–60% ↓ NO and PGI release ↑ Vascular stiffness ↓ Vessel density Hemorrhage Minor: 26–60% Impaired platelet function Severe: <3% ↓ tissue factor on endothelial cells Thromboembolic events 5–15% ↓ endothelial cell renewal with exposure of prothrombotic ECM Platelet activation ↓ tissue factor on endothelial cells Proteinuria/edema Up to 30% Podocyte dysfunction Hypothyroidism Up to 36% Thyroid cell dysfunction ↓ Vascular density Leukopenia/immune Unknown Impaired bone marrow production and cell modulation function Usually asymptomatic Skin toxicity/hand–foot Up to 42% for sorafenib Apoptosis of epidermal cells syndrome Less for other agents

Treatment Antihypertensives

Severe: hemostasis via pressure, embolization, surgical repair Anticoagulation Recommend discontinuation of most agents Hold treatment Thyroid hormone replacement

None Consider stopping if severe Avoid cold or physical activity Gloves Skin care Antibiotics for open lesions From Seiwert TY, Cohen EE. Targeting angiogenesis in head and neck cancer. Sem Oncol. 2008;35(3):274–85. Copyright Elsevier 2008

37  evaluable patients with recurrent/metastatic HNSCC, demonstrated a partial response in 1 patient, stable disease in 18 patients, and progressive disease in 19 patients, by RECIST criteria. Grade ³3 tumor bleeding occurred in 6  patients, with 2 of those patients demonstrated fatal bleeding [92]. In contrast to sorafenib and sunitinib, vandetanib is a selective dual inhibitor of EGFR and VEGF signaling. RTOG 0619, an ongoing phase II trial, randomizes 170 patients with resected locally advanced HNSCC with high-risk features to chemoradiotherapy with or ­without vandetanib [52].

Toxicities Under normal physiological circumstances, more than 99% of endothelial cells are quiescent [93, 94]. During early development, antiangiogenic therapies were anticipated to carry minimal toxicity, given the selectiveness of these agents for proliferating endothelial and perivascular cells [95, 96]. Clinical experience to date, however, has changed these expectations, with a characteristic toxicity profile now better understood. Underlying these toxicities are targeted signaling pathways critical for not just angiogenesis but other physiological processes as well. In addition, the possibility for “off-target” effects exerted by angiogenesis inhibitors on other signaling pathways complicates the picture. The most typical side effects of antiangiogenic agents involve hypertension, hemorrhagic complications, thromboembolic

events, wound healing, perforation, hypothyroidism, ­immunosuppression, proteinuria, edema, and hand–foot ­syndrome (Table 20.2) [97, 98].

Other Biologic Targets SRC Family Kinases Src family kinases consist of 8 family members, with c-Src most often overexpressed and/or aberrantly activated in epithelial and nonepithelial cancers [99]. Src kinases are associated with tumorigenesis, tumor progression, and/or metastases [100, 101]. In HNSCC, Src kinase activation follows EGFR stimulation and can be inhibited with EGFR targeting in preclinical HNSCC models [54, 102]. Dasatinib, a small-­ molecule inhibitor of Src family kinases, BCR-abl, c-Kit, and PDGFR, has been FDA-approved for chronic myeloid leukemia due to its ability to inhibit BCR-abl. Preclinical data has demonstrated the ability of dasatinib to overcome HNSCC tumor resistance to EGFR inhibition by cetuximab and to potentially resensitize resistant HNSCC cells to EGFR inhibition [103]. This has contributed to the clinical testing of dasatinib in ongoing phase II trials for recurrent/metastatic HNSCC. Preliminary results from a phase II study of dasatinib, dosed at 100 mg twice daily, in recurrent or metastatic HNSCC demonstrated fairly high rates of hospitalization (4 of 15 patients) and discontinuation (5 of 15 patients). Pharmacokinetic evaluation in this study is ongoing [104].

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The Insulin-Like Growth Gactor-1 Receptor (IGF-1R) Overexpressed in multiple human epithelial tumors, the insulin-like growth factor-1 receptor (IGF-1R) binds ligand IGF to stimulate a multitude of signaling cascades. Preclinical studies have shown that IGF-1R inhibition attenuates tumorigenesis and tumor invasion and metastasis [105]. In addition, the heterodimerizaiton of IGF-1R with EGFR may promote resistance to EGFR inhibition [106, 107]. Thus, combined targeting of IGF-1R and EGFR may be effective in enhancing antitumor activity. To address this potential, an ongoing randomized phase II trial is evaluating a monoclonal antibody of IGF-1R, IMC-A12, alone or in combination with cetuximab in patients with recurrent/metastatic HNSCC [52].

p53 The tumor suppressor gene p53 has been implicated in HNSCC, with 43% of invasive HNSCC’s harboring p53 mutations and an increased frequency of p53 mutations observed in patients with a history of tobacco use [108, 109]. A novel approach to targeted therapy, INGN 201 is a gene therapy in which a replication-defective adenovirus serotype 5 vector with a p53 combinatorial DNA insertion is administered directly into the tumor to replace mutated p53 genes with wild-type (normal) p53 genes. A phase III trial of INGN 201 compared to methotrexate in HNSCC refractory to surgery, radiotherapy, and platinum or taxane-based chemotherapy is ongoing, with the primary endpoint of overall survival. A phase II feasibility study of surgery with perioperative INGN201 gene therapy followed by chemoradiotherapy for locally advanced resectable HNSCC has recently completed accrual [52].

Summary Molecular targeted therapies for head and neck cancer offer promising opportunities to improve upon clinical outcomes of conventional treatments. Phase III clinical data demonstrate measurable survival benefits with the use of cetuximab in the locally advanced and recurrent/metastatic settings. EGFR tyrosine kinase inhibitors, erlotinib and gefitinib, and VEGF inhibitors, bevacizumab and multikinase inhibitors, have shown activity against HNSCC in a variety of settings. The toxicities of these agents are unique to their signaling cascades and, importantly, do not appear to directly overlap or amplify the toxicities of conventional treatments. The future

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impact of targeted therapies in HNSCC relies on rigorous preclinical and clinical evaluation of promising new agents and advances in our ability to predict those patients most likely to benefit.

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Chapter 21

Phase I Study Methodology in Head and Neck Oncology Christophe Le Tourneau and Lillian L. Siu

Abstract  Phase I trials evaluating the combination of radiotherapy (RT) with anticancer agents in locally advanced head and neck squamous cell carcinomas (HNSCC) require disease-specific considerations. Given that these trials are often being conducted in patients with a curative diagnosis, both safety and efficacy are relevant factors in their design and conduct. Preclinical evidence of safety, as well as appropriate biological justification of antitumor activity, should be available to rationalize the incorporation of new agents in the combination treatment regime. Anticancer agents are typically given in combination with RT for a limited amount of time and their toxicity profile has usually been well described when used without RT. However, late toxicities can occur with RT, thus the risk of exacerbating such toxicities with the addition of a systemic agent needs to be considered. For this reason, the duration of time allocated for assessment and clearance of dose-limiting toxicities typically differs from phase I trials of systemic agents without RT. A balance must be strike to optimize patient safety while maintaining a feasible timeframe for trial completion. Innovative clinical trial designs, as well as the identification of valid predictive biomarkers of toxicity and antitumor activity, are urgently needed to expedite the development of safe and effective combination regimens in locally advanced HNSCC. Keywords  Phase I trial • Dose escalation • Dose-limiting toxicity • Novel agents • Radiotherapy

Introduction Phase I clinical trials aim to establish the recommended dose or schedule of a new intervention. In oncology, these trials have primarily been designed to evaluate the safety of new

L.L. Siu (*) Division of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, ON, Canada e-mail: [email protected]

cytotoxic anticancer agents in successive cohorts of cancer patients treated with increasing doses until dose-limiting toxicity is observed in a prespecified proportion of accrued subjects. The design and methodology of phase I trials comprise of many components, including the choice of the starting dose, target toxicity level, number of patients per dose level, dose escalation method, specification of dose-limiting toxicity, and definition of the maximum tolerated dose and recommended phase II dose. As efficacy is usually not the primary endpoint of phase I cancer clinical trials, these studies are often performed in unselected tumor types. However, phase I trials specific for patients with head and neck squamous cell carcinomas (HNSCC) have emerged in the 1990s, and several reasons might explain this shift. First, HNSCC treatment in the current era involves complex multidisciplinary strategies including surgery, radiotherapy (RT), chemotherapy, and molecularly targeted agents. These strategies may therefore produce unexpected treatment-related toxicities due to interactive effects and need to be exclusively assessed in the HNSCC population, as extrapolation from other tumor types may not be appropriate. Second, RT alone, or in combination with drugs, is a definitive treatment modality delivered with a curative intent in localized or locally advanced nonmetastatic HNSCC. As such, the evaluation of efficacy in addition to toxicity is of relevance when combining RT with new drugs. Third, molecularly targeted agents that propose to modulate specific targets in tumor cells are assumed to exert disease-specific anticancer activity. For instance, the epidermal growth factor receptor (EGFR) pathway plays a more critical biological role in HNSCC than in certain other solid tumors. Fourth, the prevalence of comorbidities in some HNSCC patients may hamper the delivery of a new intervention using dosing schedules defined for other malignancies. Fifth, adverse effects on mucosa and skin rendered by new interventions may be unacceptably exacerbated in patients suffering from locoregional recurrence of HNSCC due to potential prior therapy with surgery and/or RT. Lastly, late and delayed toxicities are important in HNSCC as they are frequently associated with functional impairment of vital organs.

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_21, © Springer Science+Business Media, LLC 2011

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The development of combination strategies in HNSCC that utilize multiple therapeutic modalities continues to evolve and needs to be evaluated for safety and tolerability in phase I trials before being compared against existent standard treatments for efficacy. As phase I trial methodology involving chemotherapy and/or molecularly targeted agents without a RT component is similar across advanced solid tumors, this chapter thereby focuses on the design of phase I trials for combining RT with anticancer agents in locally advanced HNSCC (Table  21.1 and Fig.  21.1). In clinical trials enrolling this curable patient population, efficacy

Table 21.1  Considerations for phase I trials combining radiation with anticancer agents in locally advanced SCCHN Specific considerations Issues Methodologic considerations

Dose escalation methods: rule-based (e.g., standard 3+3 design) or model-based (e.g., Bayesian design) “Dose-intensity escalation” with escalation of dose of drug and/or number of drug administrations

Safety considerations

Use of preclinical models to predict safety; limitations such as the availability of appropriate models, cross-species specificity; extent to which these evaluations need to be completed prior to initiation of human phase I trial Choice of starting dose of anticancer agent to be combined with radiation Optimal length for observation for dose-limiting toxicity including acute and late toxicity Novel phase I trial designs to enable late toxicity assessment without causing delay in dose escalation Predictive markers of late toxicity

Efficacy considerations

Use of preclinical models to predict efficacy; limitations such as the availability of appropriate models, cross-species specificity; extent to which these evaluations need to be completed prior to initiation of human phase I trial Efficacy of anticancer agent to be combined with radiation Maintenance of radiation dose intensity in phase I trial Compliance of anticancer agent and of radiation therapy Novel phase I trial designs to account for efficacy in addition to toxicity Identification of surrogate markers of efficacy

Special considerations

Challenges for drug combinations to add to definitive radiation therapy Possibility of development of platinum-free radiation combinations with new drugs Phase I trials with a neoadjuvant chemotherapy component to add to chemoradiation as sequential therapy

considerations are as critical as safety issues. Trials investigating multiple-drug combinations as well as those involving a neoadjuvant chemotherapy component raise specific methodological concerns and thus will also be discussed in this chapter.

Methodologic Considerations Dose Escalation Methods in Phase I Trials Combining Radiation with Anticancer Agents Most phase I dose escalation methods in oncology have been designed under the assumption that both efficacy and toxicity increase monotonically with dose. As such, the recommended phase II dose has traditionally been established as the highest safe dose, based on a prespecified acceptable level of dose-limiting toxicity. The most commonly used dose escalation method for phase I trials remains the traditional 3+3 dose escalation method, for which dose levels are prespecified, and dose increments often become smaller as the dose increases. The 3+3 method is a rule-based method that proceeds as follows: if none of the first three patients enrolled in a cohort experiences a dose-limiting toxicity, then another three patients will be treated at the next higher dose level. However, if one of the first three patients encounters a dose-limiting toxicity, then up to three more patients will be added to the same dose level. If the target toxicity level has been preset at 33% or less, then dose escalation would stop if two or more patients among a cohort of three to six patients experience doselimiting toxicity. Besides the 3+3 method, other dose escalation methods such as Bayesian designs (e.g., modified continual reassessment method) which are model-based, have been developed but under-utilized for trials involving anticancer agents without RT [1]. Bayesian models require an initial estimate of the prior distribution of the dose– toxicity relationship, and then toxicity data obtained from patients enrolled in each dose level provide additional information for the statistical model to produce the posterior distribution. The latter is then used to help identify the dose closest to the target toxicity level. No specific dose escalation methods have been proposed for trials of anticancer agents given in combination with RT, but the traditional 3+3 method or its variations are the most widely employed. Although the dose of an anticancer agent to be combined with RT would usually be escalated if tolerable to its full monotherapy recommended phase II dose, the optimal dose to combine with RT remains elusive, especially for some molecularly targeted agents which do not exhibit dose-dependent antitumor activity.

21  Phase I Study Methodology in Head and Neck Oncology

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Fig. 21.1  Selected key considerations for the design of a phase I clinical trial of drug X in combination with RT in head and neck cancer. CT chemotherapy, RT radiotherapy, DLT doselimiting toxicity, RP2D recommended phase II dose, PD pharmacodynamic, ^ if drug X is noncytotoxic

Concept of “Dose-Intensity Escalation”

Safety Considerations

One unique feature of trials involving chemoradiation is that the anticancer agent is administered for a limited period of time, primarily during RT. The total dose of the anticancer agent administered to patients during this limited period of time can be increased by (1) escalating the dose of the drug with a fixed number of drug administrations, (2) escalating the number of drug administrations with a fixed dose of the drug, and (3) escalating both the dose and the number of drug administrations. In this context, the term “doseintensity escalation” rather than “dose escalation” would more accurately reflect the different ways to increase the total administered dose of the drug. For example, a phase I trial of paclitaxel and concurrent hyperfractionated RT used a fixed dose of paclitaxel with a planned escalation of the number of administrations during RT from 3 to 6 [2]. In another phase I trial evaluating the farnesyltransferase inhibitor L-778,123 in combination with standard RT, the first dose level administered L-778,123 at the dose of 280 mg/m2/day during weeks 1, 2, 4, and 5, while the dose and the number of administrations were escalated in the second dose level to 560 mg/m2/day during weeks 1, 2, 4, 5, and 7 [3]. In this trial, dose level 2 was found to be too toxic. As such, dose level 1 was recommended for phase II evaluation, although intermediate dose levels with the escalation of either the dose or the number of drug administrations might have allowed the safe administration of a higher total dose beyond dose level 1.

Safety Issues in Phase I Trials Combining Radiation with Anticancer Agents RT and anticancer agents are combined to optimize the therapeutic index in locally advanced HNSCC. Therapeutic index is a ratio that takes into account treatment efficacy and toxicity. Efficacy in the treatment of locally advanced HNSCC is measured by the prevention of locoregional recurrence and of distant metastasis. From the perspective of toxicity, combining RT with anticancer agents not only increases acute toxicity compared to RT alone, but may also produce chronic toxicity due to delayed or cumulative adverse effects on normal tissues. Therefore, specific safety issues that warrant careful consideration in these trials include maintenance of the standard RT dose, the choice of a safe starting dose and of subsequent dose levels for the anticancer agent, and the assessment for delayed or cumulative toxicity for the combined modality therapy.

Use of Preclinical Models to Predict Safety No specific recommendations have been published on preclinical in vitro and in vivo models that reliably predict the safety of combination of RT and anticancer agents in humans. Preclinical studies may provide some useful safety data to

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guide dosing, such as therapeutic index of the RT-anticancer agent combination. However, preclinical data must be interpreted with caution due to their limited cross-species predictability. Radiation in-field toxicity in the head and neck region that may be exacerbated by the addition of a new anticancer agent is of particular significance in HNSCC. Therefore, it would seem prudent to perform preclinical toxicity evaluations of RT and new drug combinations on mucosal, salivary gland, and neural tissues [4]. The extent to which these preclinical evaluations should be completed prior to the initiation of human phase I trials remains unclear.

Choice of the Starting Dose of Anticancer Agent to Combine with RT and Dose of RT in Phase I Trials Once a new RT-anticancer agent combination is thought to be safe to enter human testing, based on properly conducted preclinical studies, the choice of the starting dose of the anticancer agent and subsequent dose escalation are key elements of the phase I trial design. While there is guidance on the choice of the starting dose of a novel anticancer agent entering phase I evaluation as monotherapy [5], no recommendations have been established to determine the starting dose of an anticancer agent to be combined with RT. Generally, the toxicity profiles of anticancer agents intended to be combined with RT would already have been described as single agents. The therapeutic index of a new combination evaluated in preclinical models should help identify a safe starting dose and schedule, along with subsequent dose escalation for phase I trials. If preclinical data indicated a wide therapeutic index for the combination, it is reasonable to use a higher starting dose along with greater dose increments between dose levels for the anticancer agent, whereas a narrow therapeutic index would stipulate a more conservative strategy. Nevertheless, the translation of available preclinical data to the clinic may not be straightforward. This was illustrated in a phase I trial that combined RT with weekly gemcitabine in locally advanced HNSCC [6]. Although the starting dose was selected based on preclinical data showing safe and potent radiosensitization at concentrations well below cytotoxic levels, only 1/30 of the initial starting dose of gemcitabine was ultimately deemed to be safe without causing excessive early as well as delayed toxicities.

Optimal Length for Dose-Limiting Toxicity Assessment A safe dose for an anticancer agent to be combined with RT is a dose that does not produce excessive acute, delayed, and cumulative toxicity. Normal tissue recovery from RT is the

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main determinant of delayed or cumulative toxicity. Delayed or cumulative toxicities are of concern in situations where patients are treated with a curative intent and many survive to suffer from such adverse effects in the long term. Preclinical studies may help identify delayed or cumulative toxicities from RT by observing the animal hosts for a sufficient time to assess for expected or unexpected adverse effects. However, the risk of toxicity exacerbation by the addition of a systemic agent may not be reliably predicted by preclinical models and needs to be carefully considered in the design of phase I trials [4]. In clinical practice, the duration of time allocated for assessment and clearance of dose-limiting toxicities to enable dose escalation or de-escalation decisions differs between phase I trials of systemic agents with or without RT. The Cancer Therapy Evaluation Program (CTEP) and the Radiation Research Program (RRP) of the National Cancer Institute suggested that toxicity assessment for dose-limiting events spans the entire RT period and up to 30 days after the completion of RT [7]. Although toxicities may still occur after 30 days from RT completion, a longer assessment period would be impractical by unreasonably lengthening the trial duration. Furthermore, it is expected that at the end of a phase I trial of novel agent and RT, all adverse events including those that occurred outside of the assessment window for dose-limiting toxicity will be reviewed, to derive at a safe recommended phase II dose for subsequent evaluation.

Accounting for Delayed or Cumulative Toxicity in the Dose Escalation The suggested assessment period for toxicity of 30 days after the completion of RT in phase I trials combining RT with anticancer agents results in prolonged delays between cohort openings and closures. To avoid this limitation, several model-based dose escalation designs have been proposed that do not mandate trial suspension while patients are being observed. These designs use time-to-event endpoints. Cheung and Chappell developed a Bayesian-based method, known as the time-to-event continual reassessment method or TITECRM, which incorporates the time to the event (the event being toxicity) for each patient [8]. Simulations suggest that for treatments with late-onset toxicity, the TITE-CRM is more efficient than the traditional 3+3 design or the standard continual reassessment method for determining the maximum tolerated dose and leads to shorter trial durations [9]. Nevertheless, in two clinical trials combining RT with an anticancer agent in pancreatic cancer, this design led to the accrual of more patients to dose levels below the recommended phase II dose than was expected with the traditional 3+3 design [10, 11]. A variation of the TITE-CRM has been

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proposed in which accrual is temporarily suspended if the risk of toxicity at proposed doses for future patients is unacceptably high [12]. Although these methods may theoretically shorten trial duration in case of delayed or cumulative toxicities, they need to be optimized for successful practical application in phase I clinical trials combining RT with anticancer agents. Recently, a “rolling trials” strategy has been proposed to expedite the investigation of novel agent-RT combinations while allowing sufficient time to observe for potential delayed or cumulative toxicity. This strategy simultaneously activates several novel agent-RT combination trials such that while one trial is undergoing its mandatory waiting period to clear dose-limiting toxicity, another trial can actively recruit to fill its next cohort. This approach is particularly attractive if multiple anticancer agents are available to be combined with RT and can be evaluated by the same group of experienced investigators in the disease site of interest.

Predictive Markers of Late Toxicity The identification of clinical and laboratory predictive markers for delayed or cumulative toxicity may help prevent or reduce the risk of delayed or cumulative toxicity in patients receiving RT in combination with an anticancer agent. A retrospective analysis of three chemoradiation trials performed by the Radiation Therapy Oncology Group (RTOG) found that older age, advanced T stage, larynx or hypopharynx primary site, and neck dissection were associated with an increased risk of late toxicities defined as chronic grade 3 or 4 pharyngeal/laryngeal toxicity, and/or requirement for a feeding tube >2 years after study registration, and/or treatment-related death within 3 years [13]. Besides these clinical predictive markers of chronic toxicity, there are also research efforts in progress aiming to identify laboratorybased predictive biomarkers for late adverse events [14]. For example, Li et  al. showed that high levels of pretreatment circulating transforming growth factor beta (TGF-b), a potent fibrogenic cytokine, are associated with an increased risk of adverse effects after postoperative RT for breast cancer [15]. Similar results were observed in patients receiving thoracic RT [16]. Yuan et al. also showed that specific genotypes of the TGF-b gene were associated with lower risk of radiation pneumonitis in patients with non-small cell lung cancer treated with definitive radio(chemo)therapy [17]. However, none of these clinical or biological factors are being used in clinical practice for therapeutic decisions to select patients or to change treatment regimen, probably because of their low specificity. No predictive markers specific for late toxicity in HNSCC have been reported in the literature so far.

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Efficacy Considerations Efficacy Issues in Phase I Trials Combining Radiation with Anticancer Agents Historically, the rationale for combining RT with anticancer agents has mainly been driven by pragmatic approaches rather than based on preclinical scientific evidence [18]. For most drugs given in combination with RT for HNSCC, synergism in preclinical models has usually been observed [19–21]. An example of consistency between preclinical and clinical studies includes EGFR inhibitors that have shown in both preclinical and clinical studies to produce greater activity when combined with accelerated over standard fractionation RT [20, 22]. However, some preclinical data have not been validated in the clinical setting. For example, although some preclinical studies have shown that lower doses of cisplatin tend to produce increased radiosensitization than higher doses [21, 23], the FDA-approved regimen in locally advanced HNSCC involves high dose cisplatin because of the lack of randomized trial comparing low versus high doses of cisplatin in combination with RT. As patients are often being treated with a curative intent, it is essential to minimize the likelihood of compromising their outcome while evaluating new chemoradiation combinations. The pragmatic approach is certainly not optimal, and preclinical results are now expected before launching a new RT-anticancer agent combination in the clinic.

Use of Preclinical Models to Predict Efficacy In order to maximize safe and efficient investigations of new drugs in combination with RT, the CTEP and the RRP of the National Cancer Institute have provided guidelines regarding required preclinical studies before launching new chemoradiation combinations in the clinic [7]. They recommend the demonstration of in vivo synergy with fractionated RT, with little or no radiosensitization of normal tissues in two different tumor models. Additivity or synergy between RT and anticancer agents may be achieved by the modulation of the classical 5 Rs, which comprise Radiosensitivity, DNA damage Repair, cellular Repopulation and proliferation, Reoxygenation of hypoxic tumor cells, and Redistribution from more resistant to more sensitive phases of the cell cycle. Three decades ago, Steel and Peckham proposed a method to study drug–RT interactions based on the isoeffect concept reflected via the construction of isobolograms [24]. The central concept in the isobologram method is the determination of the envelope of additivity delineated by boundaries within which all responses are deemed to be purely additive.

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These boundaries are determined by the addition of responses to each agent applied alone. Preclinical studies to evaluate tumor control or growth delay at biologically relevant doses of the anticancer agent and fractionated RT should be performed under controlled conditions (i.e., using RT alone and drug alone) [4]. The testing of multiple dosing schedules of drug and RT administration can help bring the most optimal schedules of combination to clinical trials.

Efficacy of Anticancer Agent to be Combined with RT It is unclear whether new drugs should display a minimum threshold of clinical efficacy as a single agent in recurrent and/ or metastatic HNSCC before being tested in combination with RT for locally advanced disease. Anticancer agents commonly used in combination with RT for locally advanced HNSCC, including cisplatin, carboplatin, 5-FU, and cetuximab, have demonstrated single agent efficacy in HNSCC patients. However, some new drugs are being studied in combination with RT despite lacking single agent activity. For instance, raltitrexed, an antimetabolite agent, was studied in combination with RT [25], although it was shown to display minimal antitumor activity as single agent in the inoperable setting [26]. Similarly, lapatinib, a dual EGFR and HER-2 inhibitor, was studied in combination with RT and cisplatin [27], even though no clinical activity had been observed as a single agent in recurrent and/or metastatic HNSCC [28]. These two agents have only been tested in single arm phase II trials in combination with RT. To demonstrate the relevance of this strategy, randomized controlled trials would be needed to determine whether these agents can improve patient outcome when combined with RT despite minimal single agent activity in the recurrent and/or metastatic setting. Regardless of the properties of the anticancer agent to be combined, it is imperative that the dose of RT is not compromised to allow greater tolerance of the drug. Given that locally advanced HNSCC is a curative disease, the delivery of definitive dosages of RT is critical to ensure that the therapeutic efficacy is not affected by the systemic agent being added as an adjunct. Hence, compliance with the delivery of both RT and of the anticancer agent needs to be considered in the definition of dose-limiting toxicities of a combination regimen.

Accounting for Efficacy in the Dose Escalation In phase I cancer clinical trials of novel anticancer agents, efficacy is generally not the primary endpoint. However, in trials where patients are treated with a curative intent, the evaluation of efficacy in addition to safety is relevant.

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Novel trial designs have therefore emerged attempting to define a safe recommended phase II dose while simultaneously taking into consideration antitumor activity. Bayesianbased methods have been developed that incorporate both toxicity and efficacy in their designs. These methods have been originally designed for anticancer drugs used without RT, but can readily be applied to drugs in combination with RT. The EffTox method defines an acceptable dose combination based on trade-offs between the probabilities of treatment efficacy and toxicity [29]. The TriCRM is another Bayesian-based method that considers three categories (no efficacy and no toxicity, efficacy only, and toxicity only) [30]. Some investigators have proposed methods for drug combination studies that use both toxicity and efficacy as endpoints. In the design proposed by Yin et al., patients are randomly assigned among several combinations that are selected by a statistical model to determine the most effective and least toxic combination [31]. The main issue with these methods is that they assume response that can be accurately and rapidly assessed with standard response criteria or with surrogate endpoints in order to maintain a short assessment period. For instance, the use of time-to-event endpoints such as progression-free survival at 6  months would obviously lead to unacceptably long trial delays and closures. On the other hand, the use of objective response according to RECIST criteria after the completion of chemoradiation may not be relevant, as most of patients usually respond to treatment. The use of complete response is not a valid marker of overall survival in locally advanced HNSCC, since the absence of radiological complete response does not necessarily implicate the presence of residual disease but may due to treatment effects or scar tissues. The search for validated markers or endpoints of efficacy in this patient population is therefore of paramount importance before implementing these methods in clinical practice.

Special Considerations Clinical Development Challenges In the past, locally advanced HNSCC was managed primarily with RT and/or surgery. Chemotherapy has only been used in the metastatic setting or if recurrences were not amenable to surgery or re-irradiation. Since the publication of the MACH-NC meta-analysis, the benefit of concomitant platinum-based chemoradiation over RT alone has been established in this patient population [32]. Recently, the anti-EGFR monoclonal antibody cetuximab was approved in combination with RT in locally advanced HNSCC [22], based on a large randomized trial that compared this strategy against RT alone, which represented standard of care at the time the trial

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was designed. With the shift of platinum-based chemoradiation as the current standard of care, three potential strategies can be perceived to further improve therapeutic index and clinical outcome. The first is a chemo-additive strategy by adding another drug to cisplatin. However, this strategy might lead to unacceptable toxicity even when drugs without overlapping toxicities are combined with cisplatin, as illustrated in a phase II trial combining concomitant boost RT with high dose cisplatin and cetuximab [33]. The second strategy is a chemo-sparing strategy which investigates platinum-free RT combinations with new drugs. The last strategy involves the addition of a neoadjuvant component prior to the delivery of chemoradiation.

Challenges for Drug Combinations In situations where multiple systemic agents are combined with RT, the dose escalation can be challenging and raises specific issues in the design of phase I trials. If full doses of all agents cannot be delivered safely, the selection of appropriate dose combinations and schedules of systemic agents to combine with RT is not always straightforward. Most phase I trials evaluating several anticancer agents in combination with RT escalate the dose of only one agent, keeping fixed the doses of the other(s). However, it may not be always possible to administer all drugs at their recommended phase II doses as single agents. For example, while the weekly recommended phase II doses for cisplatin and docetaxel given as single agents in combination with RT are 40 and 15 mg/m2, respectively [34], the combination of the two drugs with hyperfractionated RT showed that they could only be administered at the doses of 15 and 10 mg/m2, respectively [35]. Hence, the decision of which drug to be administered at its full dose can be challenging. Several Bayesian-based designs specific for combination trials have been developed in an attempt to minimize this uncertainty [31, 36–39]. These designs do not require any prior assumption about the best dose combination, and aim to guide the dose escalation of the agents based on all toxicities observed. The ultimate goal is to determine the most active drug combination among those deemed to be safe. These methods may determine several maximum tolerated doses, and the investigator may then choose the one with the best therapeutic index as the recommended phase II dose.

Development of Platinum-Free RT Combinations with New Drugs The investigation of platinum-free combinations with RT in locally advanced HNSCC is appealing since the platins are associated with substantial acute and long-term toxicity.

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However, since platinum-based regimens remain the standard of care in this curative setting, the aim to develop non platinumbased regimens to combine with RT can be challenging. Three approaches may be taken to develop new platinumfree chemoradiation combinations without compromising efficacy. The first approach involves the initial evaluation of new agents in combination with RT in the recurrent setting where therapeutic options remain limited. One drawback might be the difficulty to reliably extrapolate the safety profile of the investigational drug(s) from an advanced to a localized disease setting, as the latter group of patients are generally in better physical condition. This was illustrated in a phase I trial evaluating concomitant chemoradiation with bevacizumab, a monoclonal antibody targeting the vascular endothelial growth factor, in previously irradiated patients with locoregionally recurrent disease [40]. Five of 26 patients treated in the expansion cohort died, two of them from stroke and hemorrhage possibly related to bevacizumab. In contrast, chemoradiation with bevacizumab seemed to be well tolerated when used in untreated patients [41]. Furthermore, the potentially higher rate of adverse events in a heavily pretreated patient population may lead to a lower recommended phase II dose of the new agent than one that could be safely administered in untreated patients. This issue was highlighted in a phase I trial that evaluated concomitant pemetrexed and cetuximab in combination with RT in two different cohorts, depending on whether patients had received previous RT or not [42]. The recommended phase II dose for pemetrexed differed in the two cohorts, suggesting that previously irradiated patients may not tolerate the same dose intensity of concomitant systemic therapy than treatment-naïve patients. The second approach utilizes the delivery of platinumfree regimens with a curative intent concomitantly with altered fractionation instead of standard fractionation RT. This approach assumes that the incorporation of a more intensified RT schedule would compensate for any potential loss of benefit due to the administration of a platinum-free regimen. Evidence in support of this approach includes the MARCH meta-analysis which reported a survival benefit with the use of altered fractionation RT schemes compared to standard fractionation RT [43]. Furthermore, a subgroup analysis of the trial by Bonner et al. showed that the addition of cetuximab was more effective when combined with altered fractionation than standard fractionation RT [22]. In another trial, the small molecule EGFR tyrosine kinase inhibitor gefitinib was designed to combine with altered fractionation RT using a concomitant boost scheme [44]. The third approach evaluates platinum-free combinations in the context of intolerance or contraindication to platinum compounds, such as in patients over 70 years of age who do not appear to derive benefit from the addition of concomitant

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platinum to definitive RT [45]. However, the accrual of these selected patient populations to complete large randomized trials may be difficult due to their relative infrequent incidences in clinical practice. As well, the incorporation of new agents may be compromised in patients with renal dysfunction. Lastly, the results that can be generated by these trials would guide management for these subgroups but are not generalizable to patients who can tolerate platinum-based regimens.

Phase I Trials with a Neoadjuvant Chemotherapy Component A survival benefit has been shown by the addition of a taxane to neoadjuvant chemotherapy with cisplatin and 5-FU, compared to cisplatin and 5-FU alone, before delivering definitive RT-based treatment for locally advanced HNSCC [46, 47]. Phase I clinical trials evaluating these so-called sequential strategies, which consist of neoadjuvant chemotherapy plus concurrent chemoradiotherapy, have become prevalent. Such trials can theoretically proceed with the dose escalation of one or several anticancer agents in either the neoadjuvant chemotherapy component, or the concurrent chemoradiotherapy component, or both. In addition to the safety issues described above when RT is combined with anticancer agents without a neoadjuvant component, phase I trials incorporating an additional neoadjuvant chemotherapy component may further influence patients safety. First, there may be potential cumulative toxicities of drugs when used in both the neoadjuvant and concurrent components. For instance, administering cisplatin in both the neoadjuvant and concurrent components may increase the risk of ototoxicity, neurotoxicity, and nephrotoxicity. Combinations with cisplatin and/or a taxane in the neoadjuvant setting may also lead to cumulative peripheral neuropathy. Docetaxel is thought to enhance radiation activity by inducing an accumulation of tumor cells in the more radiosensitive G2/M phase of the cell cycle [48], supporting its role in the neoadjuvant setting. However, myelotoxicity is a major toxicity of docetaxel, which may not only delay the delivery of concurrent chemoradiotherapy, but may also lead to a higher risk of myelosuppression during concurrent chemoradiotherapy by affecting the bone marrow reserve. Similarly, the use of drugs such as 5-FU in the neoadjuvant component may induce mucositis, and therefore may delay the start of concurrent chemoradiotherapy or lead to more frequent RT interruptions because of preexisting mucositis. Furthermore, it was recently reported that only 45% of patients could complete concomitant chemoradiation with high dose cisplatin after three cycles of neoadjuvant chemotherapy with cisplatin, docetaxel, and 5-FU [49]. The doseintensity of chemotherapy during chemoradiotherapy might

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be decreased in comparison to what patients could have received if no neoadjuvant component was added. As such, even if dose escalation is applied only to one component of sequential therapy, dose-limiting toxicity should be defined based on both the neoadjuvant and concurrent components, including the maintenance of RT dose intensity, in order to assess the safety and tolerability of the entire multistep treatment regimen.

Conclusions The addition of novel anticancer agents to RT-based treatment in locally advanced HNSCC aims to optimize the therapeutic index. While preclinical studies may contribute important data for the evaluation of new combinations, phase I clinical trials in locally advanced HNSCC represent a critical step during which both safety and efficacy endpoints are measured in this curative patient population. The unique aspects of these phase I trials, such as the potential for chronic toxicities related to RT and the relevance of preserving therapeutic efficacy, as well as the challenge in determining the optimal anticancer agent dose and/or schedule to be combined with RT, provide a strong impetus for the development and implementation of novel phase I trials designs in this setting.

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Chapter 22

Evidence-Based Head and Neck Oncology: Principles and Pitfalls Søren M. Bentzen

Abstract  Evidence-based Head and Neck Oncology is the implementation of, or the move toward, Evidence-based medicine (EBM) in the care of individual patients with head and neck squamous cell carcinoma. While the general principles and pitfalls of EBM apply in this subfield as well as in medicine in general, this chapter maintains a head and neck focus. A number of recent issues in head and neck oncology exemplify the wider challenges of introducing EBM in a diverse population of patients across a range of health care systems. Among the issues reviewed are early clinical trials of radiation therapy alone or combined with other modalities, randomized phase II trials, meta-analysis, efficacy endpoints in head and neck oncology, and patient reported outcomes. The likely prevalence of false-positive trial findings in the literature is discussed. This problem is partly caused by multiple comparisons and is aggravated by discordance between planned analyses according to trial protocols and those published in the literature. Finally, challenges related to incorporation of biomarkers in clinical trials are briefly summarized. Keywords  Head and neck oncology • Biostatistics • Trial design • Endpoints • Evidence-based medicine • Clinical research methodology

Evidence-Based Medicine: A Short Introduction Following Sackett [1], Evidence-based Medicine (EBM) is the “conscientious, explicit and judicious use of the current best evidence in making decisions about the care of individual patients.”

S.M. Bentzen (*) Department of Human Oncology, Medical Physics, and Biostatistics and Medical Informatics, University of Wisconsin – Madison, School of Medicine and Public Health, K4/316 Clinical Science Center, 600 Highland Avenue, Madison, WI 53792, USA e-mail: [email protected]

Eddy has argued that this definition is centered on the ­physician as the sole, or at least the main, provider of care and that the definition in fact focuses on Evidence-based Individual Decision making (EBID). He suggests that the development of Evidence-Based Guidelines (EBG), also known as Evidence-based Health Care, should be considered an equally worthy but separate branch of EBM. While Eddy’s distinction makes sense, it still appears that EBG is a means to an end, namely the use of current best evidence in decision making at the individual patient level. In this chapter, Sackett’s definition will be used in a broad sense, encompassing all decisions involved in caring for an individual patient, whether or not these are supported by EBG. Evidence-based medicine is a compelling concept: the idea is that all medical interventions should be supported by evidence for a net benefit for the patient over that of other therapies. The overall aims of EBM are to improve treatment efficacy and safety, to optimize health-related quality of life (QoL) for the individual patient and to reduce health care costs. In the early 1990s, an expert committee of the US Institute of Medicine estimated that only 4% of all medical services were supported by strong evidence and more than 50% had very weak or no evidence. More recent estimates are not available and whether these figures have substantially changed in the last 15  years is anybody’s guess. Several ambitious EBM initiatives have been launched in the meantime. A major driver of EBM has been the not-for-profit Cochrane Collaboration, a large network of academic collaborators committed to preparing and disseminating systematic reviews of health care [2]. In addition, Cochrane collaborators have conducted and published a number of very helpful studies on research methodology and critical reviews of the quality of the literature underlying EBM. Comparisons have shown that Cochrane reviews have greater methodological rigor and are more frequently updated than other systematic reviews and meta-analyses [3]. A critic would point out that the methodological aspects on which Cochrane reviews score highly are exactly those that, although admittedly quite reasonable, are prioritized by the Cochrane collaborators! Independent “reviews of reviews”

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have found that even if Cochrane reviews tend to follow more rigorous quality criteria, there is still room for improvement: a comparison of systematic reviews of critical care topics [4] found that adequate discussion of the quality of the included trials was missing in 44 and 79% of the Cochrane versus other systematic reviews, respectively. Several countries have national bodies aiming to advance EBM. In the US, the Agency for Healthcare Research and Quality under the Department of Health and Human Services has established an Evidence-based Practice Center program with the aim of performing systematic reviews of best available evidence for clinical and health care policy decision making [5]. In the UK, the National Institute for Clinical Excellence [6] was founded in 1999 by the National Health Service (NHS). The name was changed in 2005 after a merger with the Health Development Agency to the National Institute for Health and Clinical Excellence, although the acronym NICE was maintained. NICE recommendations are legally binding for the individual NHS trust. This has been the cause of much controversy when individual patients or various patient interest groups have felt that NICE recommendations have been unreasonably restrictive regarding access to novel therapies [6]. While some of the criticism has related directly to how NICE functions, other ­criticism seems to be misguided and should more properly have been aimed at the health policy makers. Evidence-based Head and Neck Oncology (EBHNO) is the implementation of, or the move towards, EBM in the care of individual patients with head and neck squamous cell carcinoma (HNSCC). While the general principles and pitfalls of EBM apply in this subfield as well as in medicine in general, the current chapter will attempt to maintain a head and neck focus. A number of recent issues in head and neck oncology exemplify the wider challenges of introducing EBM in a diverse population of patients across a range of health care systems.

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an associated confidence interval. The magnitude of the effect estimate does not in itself provide an indication of how strong the evidence for a benefit from one of the trial arms is. It is necessary also to consider the width of the 95% confidence interval. Multiple effect estimates can be pooled in a meta-analysis, providing an overall best estimate, see also Meta-Analysis and EBHNO Section. The other “Q” is equally important but unfortunately slightly more elusive: Trial Quality is difficult to assess objectively and even more difficult to rank on a grading scale. Different quality grading instruments have been shown to give discordant ranking of trials. It is possible to use quality as a weighting factor for example in a meta-analysis, i.e., giving relatively more weight to high-quality trials; while this is conceptually attractive it is clearly counter-indicated by the lack of an evidence-supported quality instrument. For an introductory discussion of these issues, see Bentzen [7].

Sample Size and Statistical Power A quick MEDLINE search for phase III trials in “head and neck” + “cancer” published between 2006 and 2008 produces 67 hits of which 31 are primary trial reports. Figure  22.1 shows the cumulative distribution function of sample sizes in these 31 trials. The median sample size was 163 patients, or 82 per trial arm in a 2-arm study. Using standard statistical design parameters, significance level a = 0.05 and power 1−b = 0.9, and assuming the baseline success rate to be 40%, the resolvable improvement in outcome is estimated using the arcsine approximation to be 65%, or in other words, a

Levels of Evidence The first hurdle in implementing EBM is that not all published trials of a specific intervention will reach the same conclusion regarding its risk: benefit ratio. There are two aspects to be considered: Quantity and Quality. Quantity refers to the numerical outcome estimates. Trials typically report several efficacy endpoints such as time to progression, and disease-free and overall survival. Reports on phase III trials may – in addition to significance tests for an observed difference in outcome – often include the estimated ratio of hazard rates, or hazard ratio, between trial arms. The point is that all of these effect measures are estimates derived from the observed trial outcome; they have a magnitude as well as

Fig. 22.1  Cumulative frequency distribution of the total sample size of HNSCC phase II and III trials registered in Medline 2006–2008. The upper horizontal scale indicates the minimum improvement in outcome that a trial of a given size can resolve with a significance level of 5 and 90% power. The baseline success rate is assumed to be 40%

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25% points improvement in the outcome parameter of interest. Such an improvement would be a major treatment breakthrough and it appears that most HNSCC phase III trials are under-powered to detect less spectacular but more realistic improvements of clinical outcome. Under-powered trials may lead to false-negative findings, that is, trials that on the surface seems to contradict the findings of other trials. This again leads to a lack of consistency in the clinical development of new therapies. A similar search for randomized phase II trials in head and neck cancer published 2006–2008 produced 33 hits of which 14 were primary trial reports. Figure 22.1 shows the cumulative distribution of sample sizes in these trials as well. Interestingly, two of these trials are larger than the median sample size for reported phase III HNSCC trials in the same period. However, the median sample size of the randomized phase II trials was 62 patients, which under the same assumptions as above would allow detection of an improvement in success rate from 40 to 79%, which would be nothing short of a miracle in terms of therapeutic improvement. Randomized phase II trials were originally proposed by Simon and colleagues [8] as a strategy to pick the likely best treatment out of two or more alternatives. They were not, and are not, meant to be powered for comparative effect assessment. The problem is, however, that many investigators feel that the randomization in itself justifies a formal comparison of the efficacy of treatment arms [9]. Thus, all except one of the 14 randomized phase II trial reports surveyed here included at least one P-value relating to a comparison of efficacy in the two arms of the trial. And in 8 of these 13 reports, at least one of the reported P-values comparing treatment effect was significant at the 5% level!

False-Positive Results If false-negative outcomes of under-powered trials are a concern, how can the high prevalence of trials reporting significant P-values be explained? This apparent paradox is likely explained by observing that many significant P-values are in fact false positive. Analysis of multiple endpoints, subgroup analyses, multiple looks at the data in the form of unplanned interim analyses, and early stopping of trials showing a significant benefit of one of the trial arms, all lead to a high risk of false-positive findings. Tannock [10] conducted a literature review of the actual and inferred number of significance tests comparing treatments in clinical trial reports and found that the median number of reported tests for major outcome parameters was six and estimated that the median number of reported plus implied tests was 13. If subgroup analyses were counted as well, the number of significance tests comparing treatments rose to 20. Conducting a test at the 5% ­significance

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level means that there is a 5% chance of reporting a ­significant difference even if in reality there is no difference between treatments. This nominal 5% false-positive rate will increase to 26, 49, and 64% in the case of 6, 13, or 20 significance tests even if the null hypothesis is true.

The Prevalence of False-Positive Trial Results Among all the published P-values in the literature that are significant at the 5% level, a large number are false positive. This is the background for the attention-catching title, “Why Most Published Research Findings Are False,” of John Ioannidis’ 2005 essay in PLoS Medicine [11]. The proportion of false-positive trials depends on the prevalence of truepositive treatment strategies that are tested in these. It is impossible to assess the true-positive rate, but it is likely to be quite low. In a 2005 analysis of 57 randomized controlled trials in various disease sites conducted by the Radiation Therapy Oncology Group (RTOG) between 1968 and 2002, the odds ratio for survival was 1.01 (99% CI 0.96–1.07; P = 0.5) suggesting that on average experimental and control arm therapies were equally successful [12]. If the true prevalence of improved therapies tested in trials is, say, 10%, and if the trial is designed to have the fairly standard 90% power, then 9 out of a hundred trials will come out true positive. Based on Tannock’s estimates for the number of therapy comparisons per trial (see False-Positive Results section), this translates into 72, 83, and 86% of all significant P-values being false positive in the case of 6, 13, or 20 significance tests. And remember that Tannock estimated the median number of significance tests, the total body of trial reports with significant P-values will be enriched with papers conducting more than the median number of tests. These are disturbing numbers. It is useful to look at other assumptions for the true positive proportion to see just how big a problem this is – it is BIG! Repeating “successful” trials is the remedy against falsepositive findings. This is especially effective if the real significance level is 5%, the likelihood of an ineffective therapy coming out significantly twice by chance is only 1:400. Unfortunately, in practice, both trials will typically be subject to the “false-positive” problem, in which case repeatability becomes much less of a reassurance.

Trial Registration and Public Access to Trial Protocols A balanced assessment of the efficacy of an intervention on the basis of reports in the literature is further complicated by publication bias, the well-documented fact that trials with a

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significant P-value (true or false positive) are more likely to be published than trials without a significant P-value [13, 14]. In 2005, the International Committee of Medical Journal Editors (ICMJE) initiated a policy requiring investigators to register design details of new trials into an accepted clinical trials registry before opening the trial. This was nearly 20  years after Simes had convincingly argued the case for such a register [15]. ClinicalTrials.gov, the largest trial registry in 2005, contained 13,153 trials at the time. In April 2007, the registry contained over 40,000 trials [16]. The idea is that a complete picture of the “negative” studies can only be obtained if all trials are prospectively registered before any trial outcome data become available. While the existence of these trial registries is definitely a step forward, it is also clear that registration in itself is not enough. Registered trials with a statistically significant finding are still more likely to be published than other trials and a recent review found that trials sponsored by clinical collaborative groups published 59% of registered studies, compared to a meager 5.9% of studies sponsored by industry [17]. Even when trials are published and even if the trial report concentrates on a single primary endpoint, thereby apparently minimizing the issues with multiple comparisons discussed above, there is another source of biased reporting: discrepancies between trial reports and trial protocols. Chan et al. [18] found discordance between the published primary analysis and that specified in the protocol in 25 of 42 pairs (60%) of protocols/publications. Chan also found discrepancies between protocols and publications with respect to sample size calculations (18/34 trials), methods of handling protocol deviations (19/43), missing data (39/49), subgroup analyses (25/25), and adjusted analyses (23/28). Thirteen protocols described planned interim analyses, but any such analysis was mentioned in only five of the resulting publications. It seems that the remedy against this problem is prospective registration of the full protocol and making this available for peer reviewers of the final trial publication.

Endpoints for Outcome Assessment Traditionally, HNSCC has been viewed predominantly as a locoregional disease and the main efficacy endpoint has been locoregional tumor control. Local control has been shown to be a surrogate endpoint for overall survival in trials of altered radiation dose fractionation [19]. Treatment intensity limiting toxicities have historically been localized to the highdose region as well. With a flurry of new cytotoxic or molecular targeted agents in the pipeline and new biological rationales for combining drugs with radiation also in head and neck oncology [20, 21], both efficacy and toxicity endpoints have come under scrutiny. Empirically, combinations

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of chemotherapy and radiation therapy are gaining wide acceptance as the standard of care in many patients with HNSCC. Bentzen et al. [22] identified five primary exploitable mechanisms for the rational combination of drugs with fractionated radiation therapy: spatial cooperation, cytotoxic enhancement, biological cooperation, temporal modulation, and normal tissue protection. The specific rationale exploited by a drug-radiation combination will guide the selection of optimal efficacy endpoints, facilitating a test of the underlying hypothesis for the combination.

Efficacy Endpoints The time-honored local therapy-specific efficacy endpoint in HNSCC is local, or locoregional, tumor control. At least three current trends have put a question mark over local control as the default primary endpoint in HNSCC (1) As intensified multimodality therapeutic approaches have succeeded in improving locoregional outcome, distant disease progression has become a relatively more important competing cause of treatment failure; (2) Patients having persistent locoregional disease at the end of primary treatment have local failure at time zero, but may in fact have a therapeutic benefit in terms of a prolonged time to progression from local or systemic therapies; (3) Local control is a “nonevent,” namely the absence of local failure. In some cases, there is a differential diagnostic problem in distinguishing persistent local disease from radiation sequelae. All of these considerations have turned the interest more in the direction of disease-progression based efficacy endpoints. Table 22.1 shows a proposed set of definitions for efficacy endpoints in HNSCC trials. The exact criteria for calling a progression (e.g., RECIST [23–25]) and the diagnostic procedures used to screen for progression should be defined in each case. For endpoints involving survival, death from any cause should be treated as an event. In all other cases, death will censor the observation. For a specific type of failure, say, local progression, nodal and distant progression will be ignored, i.e., the patient will still be at risk for failing locally. The exception is when patients are not followed beyond the time of the first disease progression, in which case progression outside the site of interest is a censoring event. For time to progression endpoints this author suggested to refer to the progressionfree estimate at a specific follow-up time, typically 2 or 5 years, as the progression-free rate [26]. This is not an optimal terminology as “rate” normally refers to events per unit time. Instead the term “local-progression free estimate at x years” is preferable. In randomized trials, the date of randomization is the starting time, in other types of study the first day of active therapy is generally chosen as the starting time for calculating time to failure.

22  Evidence-Based Head and Neck Oncology Table 22.1  Suggested definitions of clinical endpoints in HNSCC

Endpoint

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Abbreviation

Local progression

Nodal progression

Time to progression TTP E E Progression-free survival PFS E E Time to local progression TTLP E I Local-progression-free survival LPFS E I Time to locoregional progression TTLRP E E Locoregional progression-free LRPFS E E survival Time to distant progression TTDP I I Distant-progression-free survival DPFS I I E Event; C Censor; I Ignore. Loss to follow up is censored in all cases

Toxicity The incidence and severity of toxicity in nonsurgically ­managed HNSCC patients have increased substantially over the last two decades [27, 28]. Disturbingly, there are reasons to believe that toxicity is generally under-recorded in clinical trials and under-reported in the published literature [29–32]. Even with high-quality evidence from clinical trials, issues remain regarding the generalizability of toxicity experienced in trials to unselected patient populations treated in routine practice. New therapies are most often tested in specific populations using strict inclusion and exclusion criteria. Exclusion criteria typically comprise various types of comorbidity or lab test values outside a defined range. It has been documented that tumor outcomes in trials may overestimate the benefit from introducing experimental therapies in routine care [7]. Although less well-documented, there are good reasons to believe that trial populations may experience less toxicity than unselected clinical cases [33]. This may in part be a result of more comorbidity and a higher proportion of vulnerable patients such as the elderly and because supportive care and surveillance is more intensive in the trial setting. The incidence of events reported in clinical trials could therefore be considered the minimum estimates of risk. As clinical trials are often not powered to provide precise risk estimates, 95% confidence intervals around the estimates should be reported. This is especially an issue with late effects incidence estimates, where competing risks and incomplete follow-up may lead to an overall low number of events that again may limit the precision of these.

Patient Reported Outcomes (PRO) A strong case has been made that appropriate evaluation of new treatment strategies should incorporate the patient’s perspective [34]. Patient self-reporting has long been accepted as the preferred method for collecting QoL data and for assessing pain relief in palliative studies [35, 36].

Distant progression

Death of any cause

E E I I I I

C E C E C E

E E

C E

Patient Reported Normal Tissue Outcomes (PROs) ­comprise subjective symptoms and Quality Life (QoL). Generally speaking, there is rather poor concordance between physician and patient reported normal tissue outcomes [37–39]. Depending on the point of view, this can be interpreted either as an indication that physicians are not very good at assessing what really matters to the patient, or, alternatively, that patients are not very good at assessing their own side-effects of therapy. A more fruitful way to look at these discrepancies is to see these outcomes as complementary and at least partly independent domains of toxicity. Two recent studies provide relevant data for this discussion from slightly different perspectives. Ataman et al. [40] conducted a large study of the value of routine follow-up of cancer patients after therapy in 15 European centers, matching questionnaires filled out by physicians and patients in 2,303 cases. One of the motivations for this study was to find out if patient-initiated could replace routine follow-up visits. As part of the study, the investigators determined the proportion of patients who had a treatment-related symptom that was associated with a positive finding on clinical examination. Reanalyzing the data from the paper shows that the sensitivity of symptoms as a screen for objective clinical side-effects was 80% and the specificity was 50%. The low specificity reflects a high proportion of false positives, i.e., patients reporting symptoms but with no objective clinical signs of side-effects. In an interesting study, Basch et al. [41] used a CTCAE 3.0-based questionnaire “translated” into layman’s terms [42] and compared self- with clinician-assessment of 11 common symptoms in 400 patients and concluded that “… patients with cancer and their clinicians generally agree on the severity of symptom grades for 11 common CTCAE items.” Again, this conclusion depends on the perspective taken. Basch et al. compared discrepancies between paired patient and clinician grading, where one of the two was G0–2 and the other G3+, often regarded the threshold for a “…clinically meaningful change in management” [41] and concluded that this was seen in “fewer than 10% of all pairs.” However, 10% would correspond to some 400 pairs, or approximately one toxicity-item pair per patient in the study.

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These authors found perfect agreement in scores for between 41 and 96% of the patient–clinician pairs, depending on the item considered. However, interpretation of these frequencies is hampered by the fact that the proportion of patients with G0 score for a given item (i.e., no symptom at all), where a perfect agreement would seem more likely, is not stated in the paper. One philosophical issue is that an individual patient is an “N of 1” trial, the patient’s experience of symptoms and how they affect QoL is unique to that individual and most patients will not have an accurate impression of the spectrum of severities of side effects experienced by their fellow patients after identical treatments. While there is no discussion that the patient’s perspective needs to be further emphasized, physician assessed outcome remains an important component of recording toxicity.

How to Quantify “Therapeutic Ratio” in Oncology? Although the terms “therapeutic ratio” or “risk-benefit ratio” are widely used, there is still no consensus on how or even if, these can be meaningfully quantified [43]. Most of the methods explored in the literature require a more or less direct trade-off between toxicity and efficacy events. While this may be relatively uncontroversial in case of treatment-related mortality, the problem arises with toxicities that are not fatal or generally regarded “unacceptable.” At the individual patient level, this trade-off should clearly involve the patient’s priorities as well as a medical evaluation of risks and benefits. Informing this process requires reliable efficacy and toxicity estimates and a detailed knowledge of the toxicity profile of the therapy in question. At the societal or EBM level, there is currently no satisfactory way of quantifying the therapeutic ratio.

Specific Trial Designs Clinical trials for drug development have traditionally been conducted in three phases. A phase I trial aims to establish the recommended dose for phase II trials of a new agent or combination of agents in humans based on the occurrence of unacceptable or dose-limiting toxicity as the dose is escalated in predefined steps. Phase II trials screen a new drug for activity in a specific tumor type. Historically, phase II trials were designed to reject drugs for further consideration if they did not show a minimum clinical efficacy, typically assessed by radiological tumor regression. Drugs that show promising activity in phase II are then taken into a ­randomized

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controlled phase III trial, where the aim is to compare the new treatment with the current standard therapy. Investigators conducting radiation therapy trials have tried hard to apply the phase I, II, III model also to clinical radiation research [44]; however, local therapies and in particular radiation therapy do not fit comfortably under this research strategy.

Early Clinical Trials Involving Radiation Therapy In phase I studies, the problem is that the dose-limiting toxicities after radiation therapy are typically late effects. This is because most radiation effects are seen locally, in the highdose volume, and even relatively severe early reactions are often not dose limiting provided that the volume can be minimized. Late effects require prolonged observation of the patient and this effectively precludes rapid dose titration designs in early radiation therapy trials. Although methods have been devised [45] for pro-rating late effects according to the number of person-years at risk, which in principle will allow an early estimation of the level of late effects, these still require a sufficient number of patients with extended follow-up to be convincing. Also the standard phase II trial design is problematic; in radiation therapy, the main clinical endpoint is persistent local tumor control, whereas volume regression is not a very useful indicator of biological activity, simply because most solid malignancies regress after curative radiation doses. Most early trials of combined drug and radiation therapy do not escalate the radiotherapy component. Drug doses will be escalated using the standard 3+3 design or rapid intrapatient escalation designs [46] and the stopping rules will be defined in terms of early toxicity only. Often, there are phase I data for the drug as a sole agent or in combination with other drugs, and they are useful starting points for generating a relatively rapid escalation scheme when adding this to radiation. Due to the differences in toxicity between various organs and tissues, phase I trials of the combination must be conducted in the relevant tumor type. While phase I drug trials are often conducted in patients with a mix of tumor histologies and with no curative therapy options left, combined chemotherapy and radiation therapy phase I studies need to be conducted in patients treated with curative radiation dose schedules. Once the MTD is established, the traditional phase II study is often modified into a “feasibility study,” treating an expanded cohort of patients, perhaps 50–80, with the full therapeutic package, and the aim is to obtain more detailed toxicology – ideally also a beginning indication of late toxicity – and early efficacy data. A more complete characterization of efficacy and toxicity in combined modalities including radiation will have to come from

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a phase III trial with mature follow-up data, often meaning 5 years at least for a fair proportion of the patients.

Phase III Trials Randomized controlled trials of combination therapies involving radiation are not too different from other phase III trials. Typical primary phase III endpoints, like cause-­specific and overall survival, in a disease with a relatively favorable prognosis such as HNSCC requires long-term follow-up of patients in any case. Thereby, the available observation time becomes sufficient also to provide estimates of radiotherapyspecific endpoints such as time to local progression and late side effects. There are good statistical reasons why the primary analysis of a trial should be conducted according to the intentionto-treat principle. There is, however, an issue with relatively poor compliance in many intensified radiation fractionation [47] and combined modality regimens [48]. While poor compliance may be seen as a surrogate for toxicity in itself, this creates difficulties in interpreting both efficacy and toxicity outcomes of experimental therapies and generalizing these to routine practice.

Stratification and Predictive Biomarkers Stratified randomization, aimed at ensuring that the most important prognostic factors are balanced between trial arms, has been used extensively for several decades also in HNSCC. Stratified analyses of trial outcomes have also been used, but not to a great extent. With an improved understanding of molecular and etiological characteristics of head and neck cancers, it is safe to predict that future trials will be conducted in highly selected populations. At the time of writing, the relatively favorable prognosis in HPV-related oral and  oropharyngeal HNSCC has become well established [49–51]. In view of the considerable severity of late side-effects after current chemoradiation schedules, the idea that patients with HPV-related disease may be overtreated by standard multimodality therapies is gaining wider ­support. At the time of writing HNSCC trials with HPV positivity – often in combination with other prognostic ­factors – as an eligibility ­criterion are under development by the RTOG and other cooperative research groups. From an EBM perspective, this introduces a new degree of freedom as both the therapy and the exact definition of the trial population are being varied. A separate potentially important stratification of patients into different treatment options would be according to the

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risk of various failure types. Multiplexed immunohistochemical marker signatures may be of value in this respect [52, 53]. Novel high-throughput assays, like single nucleotide polymorphism (SNP) or DNA microarrays, hold enormous ­promise both in terms of assessing the risk of side effects and tumor recurrence, but so far none of these are near routine clinical use. One problem is the lack of definitive, large validation studies of interesting candidate biomarkers that will definitively prove (or disprove) the value of specific markers. This leads down false tracks and hinders rational progress in the field [53].

Meta-Analysis and EBHNO Two high-profile, comprehensive meta-analyses of randomized controlled trials in HNSCC have been published in the last 10 years: one concerned with the possible benefit of adding cytotoxic chemotherapy to locoregional treatment [54], the other concerned with the benefit of altered radiation therapy dose-fractionation [55]. While successfully conducting this kind of large individual-patient level analyses is a major achievement in itself, the two studies illustrate brilliantly the strengths and weaknesses of this research methodology. The strength of course is the ability to weigh evidence for or against the benefit of an intervention based on – often apparently divergent – results of a large number of trials, for ­example the chemotherapy analysis included data from 93 randomized trials comprising 17,346 patients [56]. Both analyses showed a small but highly statistically significant benefit from the experimental relative to standard therapy. In case of chemotherapy there was a 4.5% absolute increase in survival at 5 years (P < 0.0001), a slightly higher 5-year benefit, 6.5% was seen after concurrent chemo-radiation therapy (P < 0.0001). However, at the same time the test for heterogeneity was highly significant (P < 0.0001) both among all trials as well as among concurrent chemo-radiation trials [56]. A significant heterogeneity test indicates that trial outcome varies beyond what can be expected by chance if the experimental therapies had all been associated with the same therapeutic gain. In other words, the very basic assumption of the meta-analysis must be rejected. Or, put differently, there seems to be information in the variation in outcome among studies. While proponents may interpret this as an indication that there is a real benefit from chemo-radiation, the question is: “Which chemo-radiation schedule?” And if the therapeutic gains vary according to the exact therapy it becomes difficult to interpret what the pooled effect estimate really means. The pooling of multiple quite variable therapies in a meta-­analysis is associated with a loss of information, and may erroneously lead to an impression that ineffective therapies are effective or, conversely, that the benefit from the most potent therapies

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is less than what it really is. A similar limitation affects the meta-analysis of altered radiotherapy dose-fractionation in HNSCC [55] where different strategies for accelerated and/ or hyperfractionated radiation therapy were grouped into broad categories, such as “accelerated with dose-reduction” or “accelerated without dose-reduction.” The problem again is that the strategies within each category cannot be assumed to be biologically equivalent, which yet again causes problems when trying to interpret the effect estimates of the metaanalysis. Furthermore, both meta-analyses could not compile sufficient data to allow an analysis of late toxicity which evidently is an important part of selecting a therapy [29, 30]. It is conceivable that some of the therapies associated with a gain in efficacy were also associated with unacceptable or stronger than usual side effects.

Concluding Remarks: Towards EBHNO Head and neck oncology is increasingly evidence based. Yet, as discussed in this chapter, a number of issues affect the quality of published evidence. The randomized controlled trial remains the gold standard for comparative effectiveness research. There are, however, areas where the randomized trial has important limitations as a research methodology. One is health technology assessment both in the diagnostic and interventional setting [57] where the sensitivity and specificity of standard clinical effect measures in most cases is too low to allow a direct test in a reasonably sized trial [58]. Another is the integration of biomarkers into stratification of patients to various therapies. While the benefit of such a strategy can easily be tested in a randomized trial, it may in practice be difficult to conduct a straightforward comparative effectiveness trial once the stratification biomarkers are convincingly validated. Meta-analysis of evidence from randomized controlled trials is a powerful tool and the outcome of these analyses is hugely influential in shaping the ­evidence-based best practice, but again it is important to be aware of its shortcomings and pitfalls. Finally, there is the huge issue of increasing toxicity in nonsurgical management of HNSCC and the general lack of adequate documentation of this toxicity even in well-conducted clinical trials. All of these issues are challenges to be met in the coming decade.

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S.M. Bentzen 3. Jadad AR, Cook DJ, Jones A, et  al. Methodology and reports of systematic reviews and meta-analyses: a comparison of Cochrane reviews with articles published in paper-based journals. JAMA. 1998;280(3):278–80. 4. Delaney A, Bagshaw SM, Ferland A, Laupland K, Manns B, Doig C. The quality of reports of critical care meta-analyses in the Cochrane Database of Systematic Reviews: an independent appraisal. Crit Care Med. 2007;35(2):589–94. 5. Atkins D, Fink K, Slutsky J. Better information for better health care: the Evidence-based Practice Center program and the Agency for Healthcare Research and Quality. Ann Intern Med. 2005;142 (12 Pt 2):1035–41. 6. Littlejohns P, Garner S, Doyle N, Macbeth F, Barnett D, Longson C. 10 years of NICE: still growing and still controversial. Lancet Oncol. 2009;10(4):417–24. 7. Bentzen SM. A user’s guide to evidence-based oncology. Eur J Cancer Suppl. 2003;1(S6):77–91. 8. Simon R, Wittes RE, Ellenberg SS. Randomized phase II clinical trials. Cancer Treat Rep. 1985;69(12):1375–81. 9. Liu PY, LeBlanc M, Desai M. False positive rates of randomized phase II designs. Control Clin Trials. 1999;20(4):343–52. 10. Tannock IF. False-positive results in clinical trials: multiple significance tests and the problem of unreported comparisons. J Natl Cancer Inst. 1996;88:206–7. 11. Ioannidis JP. Why most published research findings are false: author’s reply to Goodman and Greenland. PLoS Med. 2007;4(6):e215. 12. Soares HP, Kumar A, Daniels S, et al. Evaluation of new treatments in radiation oncology: are they better than standard treatments? JAMA. 2005;293(8):970–8. 13. Dwan K, Altman DG, Arnaiz JA, et  al. Systematic review of the empirical evidence of study publication bias and outcome reporting bias. PLoS ONE. 2008;3(8):e3081. 14. Easterbrook PJ, Berlin JA, Gopalan R, Matthews DR. Publication bias in clinical research. Lancet. 1991;337:867–72. 15. Simes RJ. Publication bias: the case for an international registry of clinical trials. J Clin Oncol. 1986;4(10):1529–41. 16. Laine C, Horton R, DeAngelis CD, et al. Clinical trial registration: looking back and moving ahead. Lancet. 2007;369(9577):1909–11. 17. Ramsey S, Scoggins J. Commentary: practicing on the tip of an information iceberg? Evidence of underpublication of registered clinical trials in oncology. Oncologist. 2008;13(9):925–9. 18. Chan AW, Hrobjartsson A, Jorgensen KJ, Gotzsche PC, Altman DG. Discrepancies in sample size calculations and data analyses reported in randomised trials: comparison of publications with protocols. BMJ. 2008;337:a2299. doi:10.1136/bmj.a2299.:a2299. 19. Wadsley JC, Bentzen SM. Investigation of relationship between change in locoregional control and change in overall survival in randomized controlled trials of modified radiotherapy in head-and-neck cancer. Int J Radiat Oncol Biol Phys. 2004;60(5):1405–9. 20. Bernier J, Bentzen SM, Vermorken JB. Molecular therapy in head and neck oncology. Nat Rev Clin Oncol. 2009;6(5):266–77. 21. Wilson GD, Bentzen SM, Harari PM. Biologic basis for combining drugs with radiation. Semin Radiat Oncol. 2006;16(1):2–9. 22. Bentzen SM, Harari PM, Bernier J. Exploitable mechanisms for combining drugs with radiation: concepts, achievements and future directions. Nat Clin Pract Oncol. 2007;4(3):172–80. 23. Eisenhauer EA, Therasse P, Bogaerts J, et  al. New response ­evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228–47. 24. Schwartz LH, Bogaerts J, Ford R, et al. Evaluation of lymph nodes with RECIST 1.1. Eur J Cancer. 2009;45(2):261–7. 25. Therasse P, Arbuck SG, Eisenhauer EA, et  al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst. 2000;92(3):205–16.

22  Evidence-Based Head and Neck Oncology 26. Bentzen SM. Towards evidence based radiation oncology: improving the design, analysis, and reporting of clinical outcome studies in radiotherapy. Radiother Oncol. 1998;46:5–18. 27. Bentzen SM, Rosenthal DI, Weymuller EA, Trotti A. Increasing toxicity in nonoperative head and neck cancer treatment: investigations and interventions. Int J Radiat Oncol Biol Phys. 2007;69 (2 Suppl):S79–82. 28. Trotti A. Toxicity in head and neck cancer: a review of trends and issues. Int J Radiat Oncol Biol Phys. 2000;47(1):1–12. 29. Bentzen SM, Trotti A. Evaluation of early and late toxicities in chemoradiation trials. J Clin Oncol. 2007;25(26):4096–103. 30. Trotti A, Bentzen SM. The need for adverse effects reporting standards in oncology clinical trials. J Clin Oncol. 2004;22(1):19–22. 31. Ioannidis JP, Lau J. Completeness of safety reporting in randomized trials: an evaluation of 7 medical areas. JAMA. 2001;285(4): 437–43. 32. Ioannidis JP. Adverse events in randomized trials: neglected, restricted, distorted, and silenced. Arch Intern Med. 2009;169(19):1737–9. 33. Hassett MJ, O’Malley AJ, Pakes JR, Newhouse JP, Earle CC. Frequency and cost of chemotherapy-related serious adverse effects in a population sample of women with breast cancer. J Natl Cancer Inst. 2006;98(16):1108–17. 34. Acquadro C, Berzon R, Dubois D, et al. Incorporating the patient’s perspective into drug development and communication: an ad hoc task force report of the Patient-Reported Outcomes (PRO) Harmonization Group meeting at the Food and Drug Administration, February 16, 2001. Value Health. 2003;6(5):522–31. 35. Chow E, Wu JS, Hoskin P, Coia LR, Bentzen SM, Blitzer PH. International consensus on palliative radiotherapy endpoints for future clinical trials in bone metastases. Radiother Oncol. 2002;64(3):275–80. 36. Madsen EL. Painful bone metastasis: efficacy of radiotherapy assessed by the patients: a randomized trial comparing 4 Gy X 6 versus 10 Gy X 2. Int J Radiat Oncol Biol Phys. 1983;9(12):1775–9. 37. Davidson SE, Burns MP, Routledge JA, Swindell R, Bentzen SM, West CM. Assessment of morbidity in carcinoma of the cervix: a comparison of the LENT SOMA scales and the Franco-Italian glossary. Radiother Oncol. 2003;69(2):195–200. 38. Jensen K, Bonde JA, Grau C. The relationship between observerbased toxicity scoring and patient assessed symptom severity after treatment for head and neck cancer. A correlative cross sectional study of the DAHANCA toxicity scoring system and the EORTC quality of life questionnaires. Radiother Oncol. 2006;78(3):298–305. 39. Fromme EK, Eilers KM, Mori M, Hsieh YC, Beer TM. How accurate is clinician reporting of chemotherapy adverse effects? A comparison with patient-reported symptoms from the Quality-of-Life Questionnaire C30. J Clin Oncol. 2004;22(17):3485–90. 40. Ataman OU, Barrett A, Davidson S, et al. Audit of effectiveness of routine follow-up clinics after radiotherapy for cancer: a report of the REACT working group of ESTRO. Radiother Oncol. 2004; 73(2):237–49. 41. Basch E, Iasonos A, McDonough T, et al. Patient versus clinician symptom reporting using the National Cancer Institute Common Terminology Criteria for Adverse Events: results of a questionnairebased study. Lancet Oncol. 2006;7(11):903–9.

325 42. Basch E, Artz D, Dulko D, et  al. Patient online self-reporting of toxicity symptoms during chemotherapy. J Clin Oncol. 2005; 23(15):3552–61. 43. Bentzen SM. Design of clinical trials in radiation oncology: saving lives, not Grays. In: Dale RG, Jones B, editors. Modelling in radiation oncology. London: British Institute of Radiology, 2007; 196–211. 44. Bentzen SM, Dorr W, Anscher MS, et  al. Normal tissue effects: reporting and analysis. Semin Radiat Oncol. 2003;13(3):189–202. 45. Cheung YK, Chappell R. Sequential designs for phase I clinical trials with late-onset toxicities. Biometrics. 2000;56(4):1177–82. 46. Simon R, Freidlin B, Rubinstein L, Arbuck SG, Collins J, Christian MC. Accelerated titration designs for phase I clinical trials in oncology. J Natl Cancer Inst. 1997;89(15):1138–47. 47. Khalil AA, Bentzen SM, Bernier J, et  al. Compliance to the prescribed dose and overall treatment time in five randomized clinical trials of altered fractionation in radiotherapy for head-and-neck carcinomas. Int J Radiat Oncol Biol Phys. 2003;55(3):568–75. 48. Bernier J, Bentzen SM. Altered fractionation and combined radiochemotherapy approaches. Pioneering new opportunities in head and neck oncology. Eur J Cancer. 2003;39(5):560–71. 49. Dahlstrand H, Dahlgren L, Lindquist D, Munck-Wikland E, Dalianis T. Presence of human papillomavirus in tonsillar cancer is a favourable prognostic factor for clinical outcome. Anticancer Res. 2004;24(3b):1829–35. 50. Ragin CC, Taioli E. Survival of squamous cell carcinoma of the head and neck in relation to human papillomavirus infection: review and meta-analysis. Int J Cancer. 2007;121(8):1813–20. 51. Lassen P, Eriksen JG, Hamilton-Dutoit S, Tramm T, Alsner J, Overgaard J. Effect of HPV-associated p16INK4A expression on response to radiotherapy and survival in squamous cell carcinoma of the head and neck. J Clin Oncol. 2009;27(12):1992–8. 52. Ataman OU, Bentzen SM, Wilson GD, et al. Molecular biomarkers and site of first recurrence after radiotherapy for head and neck cancer. Eur J Cancer. 2004;40(18):2734–41. 53. Bentzen SM. From cellular to high-throughput predictive assays in radiation oncology: challenges and opportunities. Semin Radiat Oncol. 2008;18(2):75–88. 54. Pignon JP, Bourhis J, Domenge C, Designe L. Chemotherapy added to locoregional treatment for head and neck squamous-cell carcinoma: three meta-analyses of updated individual data. MACH-NC Collaborative Group. Meta-Analysis of Chemotherapy on Head and Neck Cancer. Lancet. 2000;355(9208):949–55. 55. Bourhis J, Overgaard J, Audry H, et al. Hyperfractionated or accelerated radiotherapy in head and neck cancer: a meta-analysis. Lancet. 2006;368(9538):843–54. 56. Pignon JP, Le MA, Maillard E, Bourhis J. Meta-analysis of chemotherapy in head and neck cancer (MACH-NC): an update on 93 randomised trials and 17, 346 patients. Radiother Oncol. 2009; 92(1):4–14. 57. Bentzen SM. Randomized controlled trials in health technology assessment: overkill or overdue? Radiother Oncol. 2008;86(2): 142–7. 58. Bentzen SM. Radiation oncology health technology assessment: the best is the enemy of the good. Nat Clin Pract Oncol. 2008;5(10):563.

Chapter 23

Programs of Organ and Function Preservation Jean-Louis Lefebvre

Abstract  An intensive clinical research has been carried out over the past three decades aiming to avoid performing a total laryngectomy. Large partial open procedures or endoscopic laser CO2 surgery may be an alternative to total laryngectomy in very highly selected cases. Altered fractionated radiotherapy has proved to be more efficient than conventional radiotherapy. However, most of the research has been done by combining chemotherapy and radiotherapy. The first programs used induction chemotherapy (cisplatin and 5-fluorouracil with or without docetaxel) followed by radiotherapy in good responders. Toxicity was acceptable, neither disease control nor survivals were compromised and larynx could be preserved in at least two thirds of the cases. The second programs used concomitant chemoradiotherapy. Concurrent chemoradiotherapy provided higher larynx preservation rates but at the price of a substantial acute and late toxicity potentially compromising the larynx function. Alternating chemoradiotherapy did not increase toxicity but larynx preservation was similar to induction chemotherapy. Whether concurrent or alternating, there was no improvement of survival. The on-going third programs are assessing induction chemotherapy followed by radiotherapy with either concurrent chemotherapy or concurrent biotherapy. Keywords  Larynx • Hypopharynx • Biotherapy • Radiotherapy • Surgery

•

Chemotherapy

Introduction Surgery has been the first treatment of larynx and hypopharynx cancers. This surgery was initiated at the end of the nineteenth century for larynx cancer and quite simultaneously consisted of either partial or total laryngectomy. At the very beginning of the twentieth century radiotherapy was also J.-L. Lefebvre (*) Head and Neck Department, Centre Oscar Lambret, 3 Rue Combemale, Lille 59020, France e-mail: [email protected]

used for the treatment of laryngeal malignancies. As a result, from the start there were two major options: surgery or radiotherapy. All along the twentieth century an intensive surgical research has allowed fine-tuning the indications and techniques of the various partial surgery procedures. With time some large partial procedures have been validated for cases that were until then only amenable to a total laryngectomy. That is the case, for example, of the supracricoid partial laryngectomies. Endoscopic laser surgery has also been a major advance. But open and endoscopic partial surgeries are indicated most often for quite limited tumors. Radiotherapy techniques have also been improved (better conformation of irradiated fields to the tumor volume, modification of the fractionation). Larynx preservation has been a major advance in head and neck cancer management over the past three decades. The goal of larynx preservation is to control the disease and to maintain in place a functioning larynx. This definition of larynx preservation is only meaningful if indicated for advanced larynx and hypopharynx cancers that are, if surgery is considered, only resectable by a total laryngectomy. These cases have been until the 1980s treated by either total laryngectomy with postoperative radiotherapy if indicated or by definitive radiotherapy with surgery in reserve in case of failure. These two options have never been compared in a randomized trial that should have been the first larynx preservation program. Each option was indicated according to institutional policies. The appearance of active chemotherapy regimen in the early 1980s has had a definite impact on this discussion.

Programs with Partial Surgery Some teams have explored the reliability of supracricoid laryngectomy in selected T3 and T4 larynx cancers [1, 2]. Some other teams have extended the indications of endoscopic laser surgery to T3 or T4 larynx or hypopharynx cancers [3–7]. Both have got undisputable satisfactory results but on quite limited series. Clearly, there is a room for these indications but for highly selected cases and for very

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experienced surgical teams and are not on a large scale a real alternative to total laryngectomy. Transoral robotic surgery is under evaluation but again for early diseases.

Programs with Definitive Radiotherapy Many reports have shown the improvement of radiotherapy results thanks to a modification of the fractionation. The goal is either to increase the total dose by the means of delivering more than one fraction per day (hyperfractionated radiotherapy) or to decrease the overall treatment time (reducing the ability of tumor cell repair and repopulation). A recent meta-analysis [8] has assessed the impact of altered fractionated radiotherapy on survival. A total of 6,515 patients enrolled in 15 randomized trials were included in the analysis. There was a significant 3.4% benefit in survival at 5 years as well as there was a significant better local control. The major improvement in 5-year survival was found for hyperfractionated radiotherapy (8%). The effect of altered fractionated radiotherapy on tumor control did not differ according to the primary site. However, the impact of altered fractionation on larynx preservation is probably limited. The acute toxicity of these treatments may be a limit due to the cartilaginous structure of the larynx, due to the impact of mucositis on larynx function and due to the vulnerability of the cricoarytenoid joints.

Programs Based on Chemotherapy and Radiotherapy Programs with Induction Chemotherapy In the early 1980s, the Wayne State University team reported their experience with platinum-based induction chemotherapy and in particular with the cisplatin and 5-fluorouracil regimen. Previously, untreated patients demonstrated impressive response rates at the primary tumor site. When subsequently treated with radiotherapy good responders to induction chemotherapy appeared to be also good responders to irradiation while poor responders to induction chemotherapy were also poor responders to the subsequent irradiation [9, 10]. These reports had a tremendous impact on the daily practice and induction chemotherapy was widely used for head and neck cancer. But a large meta-analysis [11] failed to find a significant advantage of induction chemotherapy in terms of survival. However, it must be stressed that when induction

J.-L. Lefebvre

chemotherapy consisted of cisplatin and 5-fluorouracil, there was a significant 5% improvement of the 5-year survival. But the undisputable merit of induction chemotherapy has been to reopen the discussion on larynx preservation. If the discussion on a randomized comparison of radical larynx surgery versus definitive irradiation had not get the consensus between surgeons and radiation, on the contrary comparing radical surgery versus definitive irradiation in good responding patients after induction chemotherapy appeared acceptable. The first program on larynx preservation could really start.

Programs with Cisplatin and 5-Fluorouracil (PF) Induction Chemotherapy The goal was to compare total laryngectomy with neck dissection with or without postoperative irradiation versus PF induction chemotherapy followed by irradiation (keeping total laryngectomy in reserve for salvage if necessary) in good responders or by total laryngectomy with or without postoperative irradiation in poor responders. The Department of Veterans Affairs Laryngeal Cancer Study Group reported in 1991 the first randomized trial on larynx cancers [12]. Three hundred and thirty-two patients were randomly assigned to be treated by total laryngectomy or to receive two cycles of PF followed in responders (partial or complete responders) by a third cycle and irradiation or by total laryngectomy in nonresponders. In this trial 63% of the patients had a supraglottic tumor and 37% had a glottic cancer, and 57% had larynx fixity. There was no significant difference in survival between both arms (68% at 2 years). At 4 years, two thirds of the survivors in the chemotherapy arm had retained their larynx. These data were updated regularly in various meetings and these results did not vary with time. A quality of life study has been carried out on 46 survivors of this trial (25 in the surgery arm and 21 in the chemo arm). Better scores were found in the chemotherapy arm patients as regards more freedom of pain, better emotional well-being, and lower levels of depression. But surprisingly there was no correlation between quality of life scores and preservation of the speech function. The European Organization for Research and Treatment of Cancer (EORTC) published in 1996 a similar trial on hypopharynx (78%) and lateral epilarynx (22%) tumors only eligible for a total laryngectomy with partial pharyngectomy [13]. Two hundred and two patients were enrolled in this study comparing the standard treatment (surgery and postoperative irradiation) versus two or three cycles of PF followed in clinically complete responders at the primary site by irradiation or, for other patients, by the conventional treatment.

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For the 194 evaluable patients, there was no significant difference in survival, despite a notable difference in median survival favoring the experimental arm (44 months) when compared with the surgery arm (25 months). Finally, at 3 and 5 years half the survivors in the chemotherapy arm had retained a functional larynx. This trial was updated with a 10-year follow-up and these results were confirmed [14]. Of note in this trial there was a specific analysis of the impact of induction chemotherapy on tolerance and quality of the subsequent treatments. Radiation therapy was not compromised by the previous chemotherapy as well there was no unforeseen treatment interruption due to acute toxicity. Salvage surgery in poor responders had similar postoperative courses and similar quality of surgical margins when compared with patients treated in the surgical arm. The French group (GETTEC) published also in 1998 a randomized trial on larynx cancer. Patients were randomized to receive either the standard treatment (total laryngectomy) or three cycles of PF followed by irradiation in case of clinical response over 80% or by total laryngectomy in the other cases [15]. In this trial, the selection was more restrictive than in the North-American study since all tumors were classified T3 and all patients had larynx fixity while only 31% had a supraglottic tumor while 69% had glottic or transglottic tumor and all had larynx fixity. The trial was prematurely closed due to a poor accrual. The 2-year survival was significantly higher in the surgery arm (84 vs. 69%) but 15 of the 36 patients (42%) enrolled in the chemotherapy arm avoided surgery. These three trials were pooled in a subset analysis of the above-mentioned large meta-analysis [11]. It appeared that there was a nonsignificant 6% decrease in survival in the chemotherapy arms when analyzed together that was balanced by a 56% larynx preservation rate.

Programs with Docetaxel, Cisplatin, and 5-Fluorouracil (TPF) Induction Chemotherapy In 2007, two randomized trials comparing the PF induction regimen with the TPF one were simultaneously published. The TAX 323 [16] trial assessed this comparison for nonresectable tumors to be treated after the induction phase with radiotherapy alone. The TAX 324 trial [17] assessed this comparison for either resectable or nonresectable tumors to be treated thereafter with radiotherapy and concurrent weekly carboplatin. Both trials concluded that the overall survival and locoregional control were significantly higher in the TPF

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arm with a reduction as high as 30% in risk of death. This superiority of the TPF regimen was also supported by another specific meta-analysis. The TPF regimen is considered as the new standard for induction chemotherapy. The French group GORTEC published in 2009 a randomized trial comparing PF and TPF as induction chemotherapy followed by irradiation in case of response of at least 50% in larynx and hypopharynx cancers [18]. A total of 213 patients were enrolled in this study. With a median follow-up of 3 years there was no difference in terms of survival but the 3-year actuarial larynx preservation rate was 70.3% with TPF versus 57.5% with PF (p = 0.03).

Conclusions for the Programs with Induction Chemotherapy Followed by Irradiation in Good Responders • The addition of induction chemotherapy for larynx preservation did not compromise the survival when compared with upfront surgery. • Induction chemotherapy did not compromise subsequent treatment (either salvage surgery of definitive irradiation) in terms of tolerance or of efficacy. • None of the different induction chemotherapy regimens (PF or TPF) has been able to improve survival in larynx preservation programs.

Programs with Concomitant Chemoradiotherapy Concomitant chemoradiotherapy may be delivered with two different schedules. Chemotherapy may be given either during irradiation without interruption in radiotherapy (concurrent chemoradiotherapy) or alternatively with radiotherapy during the radiation protocol (alternating chemoradiotherapy). The advantage of concurrent chemoradiotherapy has been demonstrated by the meta-analysis and the administration of cisplatin at the dose of 100 mg/m² on days 1, 22, and 43 of a conventional 70 Gy irradiation has been shown as the highest advantage [11, 19]. Alternating chemoradiotherapy delivering four cycles of PF (on weeks 1, 4, 7, and 10) and three courses of radiotherapy at the dose of 20 Gy in 2 weeks (weeks 2–3, 5–6 and 8–9) for a total of 60 Gy has been reported as feasible and able to improve survival and disease control [20, 21].

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Programs with Concurrent Chemoradiotherapy The RTOG published in 2003 a large three-arm randomized trial [22]. In this trial, 547 patients were randomized to receive in one arm PF induction chemotherapy followed in responders by irradiation in another arm concurrent chemoradiotherapy (with cisplatin) or in the third arm radiotherapy alone. With a median follow-up of 3.8 years the highest 2-year larynx preservation rate was found in the concurrent arm (88%) while there was no difference in 5-year overall survival between the three arms. As regards acute toxicity, the grade 3–4 mucositis was twice higher in the concurrent arm when compared with the two others. The complication rate after salvage laryngectomy did not differ between the three arms. This trial was updated in 2006 [23] and confirmed the laryngeal preservation was of 83.6% in the concurrent arm versus 70.5% in the induction arm (p = 0.0029) and versus 65.7% in the radiotherapy arm (p = 00017) but again without any difference in overall or disease-free survivals. Of note there were twice as many noncancer-related deaths in the concurrent arm when compared with the two other arms. There was no increase in late toxicity reported for this trial. However, the RTOG reported a combined study [24] of three concurrent chemoradiotherapy arms from three randomized trials conducted by this group (RTOG 91-11, RTOG 97-03, and RTOG 99-14). The aim of this study was to assess severe toxicity that occurred in 43% of patients. Severe late toxicity was found in particular for larynx and hypopharynx cancer.

Programs with Alternating Chemoradiotherapy The EORTC published in 2009 a randomized trial comparing induction chemotherapy and alternating chemoradiotherapy in patients with advanced tumor of the larynx or hypopharynx candidates for a total laryngectomy [25]. In the induction arm, patients with a 50% or more reduction in primary tumor size after two cycles of PF received another two cycles, followed by radiotherapy (70 Gy total). In the alternating arm, a total of four cycles of PF (in weeks 1, 4, 7, and 10) were alternated with radiotherapy with 20 Gy during the three 2-week intervals between chemotherapy cycles (60 Gy total). A total of 450 patients were enrolled in this trial. With a median follow-up of 6.5 years, survival with a functional larynx was similar in sequential and alternating arms as were similar larynx preservation rates and overall or progression-free survivals. The acute toxicity was slightly lower in the alternating arm but there was no difference in late toxicity.

J.-L. Lefebvre

Conclusions for the Programs with Concomitant Chemoradiotherapy • Concurrent chemoradiotherapy provides the highest larynx preservation defined as the larynx in place. • Concurrent chemoradiotherapy generates a substantial acute toxicity. • Late toxicity after concurrent chemoradiotherapy may compromise the laryngeal function. It is important to stress that for quality of life only the preservation of a functioning larynx is meaningful. • Neither concurrent nor alternating chemoradiotherapy improves survival.

Programs with Sequential Chemoradiotherapy (I.E. Induction Chemotherapy Followed by Concurrent Chemoradiotherapy) The TAX 324 trial (PF vs. TPF before radiotherapy and concurrent weekly carboplatin) showed the feasibility of sequential chemoradiotherapy [17]. Meanwhile a randomized trial on biotherapy comparing radiotherapy alone and radiotherapy with concurrent administration of a monoclonal antibody targeting the EGFR (cetuximab) showed that this combined treatment provided a significantly higher overall survival and locoregional control than radiotherapy alone, this improvement being in the range of that observed with concurrent chemoradiotherapy but without an increased acute mucosal toxicity [26].

Programs with Sequential Chemoradiotherapy A subset analysis of the TAX 324 trial was carried out on 166 patients with larynx or hypopharynx cancer [27]. The same improvement in overall survival and progression-free survival in the TPF arm was found for this subgroup of patients as for the overall population. Among the 123 operable patients, the laryngectomy-free survival was also significantly greater. The Ann Arbor group explored larynx preservation in 36 patients with T4 larynx cancers [28]. Usually these tumors are excluded from larynx preservation trials. Patients received one cycle of PF. In case of response of at least 50% the patients received thereafter chemoradiotherapy with adjuvant PF in case of clinically complete response. The larynx preservation rate was of 58%.

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Programs with Sequential Biotherapy The GORTEC group reported in 2009 the preliminary results of a randomized phase II trial in patients with larynx or hypopharynx tumor [29]. One hundred and fifty-three eligible patients were enrolled to receive three cycles of TPF. In case of response of at least 50% they were randomized to receive either concurrent chemoradiotherapy with cisplatin or concurrent radiotherapy and cetuximab. Only 74% of patients could receive the planned induction chemotherapy protocol. After induction chemotherapy 85% of patients were theoretically fulfilled the criteria for randomization but only 115 patients could be actually randomized (mainly due to toxicity induced by induction chemotherapy). Only 45% of patient randomized in the cisplatin arm could receive the planned three cycles of cisplatin while 71% of patients randomized in the cetuximab arm could receive the full protocol. Three months after treatment there was no significant difference in larynx preservation.

Conclusions for the Programs with Sequential Chemoradiotherapy • Sequential chemoradiotherapy is potentially a new option but delivering the standard induction chemotherapy followed by the standard concurrent chemoradiotherapy generates a substantial overall toxicity. • The integration of biotherapies may overcome this concern but clearly sequential chemoradiotherapy (either with cisplatin or a biotherapy such as cetuximab) remains to be evaluated on a large phase III trial specifically designed for larynx preservation.

Discussion Larynx preservation is an important new concept that has been developed for tumors of the larynx and of the hypopharynx that can be removed only by a total laryngectomy. Extensive tumors (T4) and very infiltrative transglottic tumors are at least for the moment better controlled by an upfront total laryngectomy. If the concept of larynx preservation is nowadays considered as a validated option, the best larynx preservation protocol remains to be defined. It is noticeable that all these trials even if they were conducted with the goal of larynx preservation had different definitions of larynx preservation: from the simplest one (larynx preservation = larynx in place) to the most

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complex one (survival with a larynx free of tumor and ­without tracheotomy or feeding tube). A detailed quality of the function of the preserved larynx is often missing. A consensus should be reached in designing future trials [30] [31]. None of the larynx preservation protocols has had an impact on survival. This means that none provided a better survival that an upfront total laryngectomy. Whatever the protocol, distant metastases remain a concern. It must be underscored that these larynx preserving protocols did not compromise disease local control and survival because the surgeons performed salvage surgery. It must be kept in mind that surgery plays an important role in this research. To this extent much attention must be paid to acute and late toxicity not only for the quality of the function of the larynx but also for the feasibility and reliability of salvage surgery. Larynx preservation is a challenging approach that is permanently moving. Induction chemotherapy has the advantage of allowing adapting the subsequent treatment quite early in the treatment program. Concurrent chemoradiotherapy provides high local control but at the price of a substantial acute and late toxicity. Both has advantages and disadvantages, combining both is a logical new step in this clinical research but the balance between the induction and the concurrent phases remains to be defined. Finally, biotherapies have a role to play in this research. More than ever a multinational–multidisciplinary collaboration is requested.

References 1. Chevalier D, Laccourreye O, Brasnu D, Laccourreye H, Piquet JJ. Cricohyoidoepiglottopexy for glottic carcinoma with fixation or impaired motion of the true vocal cord: 5-year oncologic results with 112 patients. Ann Otol Rhinol Laryngol. 1997;106:364–9. 2. Lima RA, Freitas EQ, Kligerman J, et al. Near-total laryngectomy for treatment of advanced laryngeal cancer. Am J Surg. 1997;174:490–1. 3. Iro H, Waldfahrer F, Altendorf-Hofmann A, Weidenbecher M, Sauer R, Steiner W. Transoral laser surgery of supraglottic cancer: follow-up of 141 patients. Arch Otolaryngol Head Neck Surg. 1998;124(11):1245–50. 4. Motta G, Esposito E, Motta S, Tartaro G, Testa D. CO(2) laser surgery in the treatment of glottic cancer. Head Neck. 2005;27:566–74. 5. Vilaseca-Gonzalez I, Bernal-Sprekelsen M, Blanch-Alejandro JL, Moragas-Lluis M. Complications in transoral CO2 laser surgery for carcinoma of the larynx and hypopharynx. Head Neck. 2003;25: 382–8. 6. Martin A, Jäckel MC, Christiansen H, Mahmoodzada M, Kron M, Steiner W. Laryngoscope. 2008;118:398–402. 7. Rudert HH, Hoft S. Transoral carbon-dioxide laser resection of hypopharyngeal carcinoma. Eur Arch Otorhinolaryngol. 2003;260: 198–206. 8. Bourhis J, Overgaard J, Audry H, et al. Hyperfractionated or accelerated radiotherapy in head and neck cancer: a meta-analysis. Lancet. 2006;368:843–54. 9. Decker DA, Drelichman A, Jacobs J, et al. Adjuvant chemotherapy with cis-diamminodichloroplatinum II and 120-hour infusion

332 5-fluorouracil in stage III and IV squamous cell carcinoma of the head and neck. Cancer. 1983;51:1353–5. 10. Ensley JF, Jacobs JR, Weaver A, et al. Correlation between response to cis-platinum-combination chemotherapy and subsequent radiotherapy in previously untreated patients with advanced squamous cell cancers of the head and neck. Cancer. 1984;54(5):811–4. 11. Pignon JP, Bourhis J, Domenge C, Designe L. Chemotherapy added to locoregional treatment for head and neck squamous-cell carcinoma: three meta-analyses of updated individual data. MACH–NC Collaborative Group. Meta-analysis of chemotherapy on head and neck cancer. Lancet. 2000;355:949–55. 12. The Department of Veterans Affairs Laryngeal Cancer Study Group. Induction chemotherapy plus radiation compared with surgery plus radiation in patients with advanced laryngeal cancer. N Engl J Med. 1991;324:1685–90. 13. Lefebvre JL, Chevalier D, Luboinski B, Kirkpatrick A, Collette L, Sahmoud T. Larynx preservation in pyriform sinus cancer: preliminary results of a European Organization for Research and Treatment of Cancer phase III trial. EORTC Head and Neck Cancer Cooperative Group. J Natl Cancer Inst. 1996;88:890–9. 14. Lefebvre J-L, Chevalier D, Luboinski B, et al. Is laryngeal preser­ vation (LP) with induction chemotherapy (ICT) safe in the treatment of hypopharyngeal SCC? Final results of the EORTC 24891 trial. J Clin Oncol. 2004 ASCO Annual Meeting Proc. 2004; 22:abstract 5531. 15. Richard JM, Sancho-Garnier H, Pessey JJ, et al. Randomized trial of induction chemotherapy in larynx carcinoma. Oral Oncol. 1998;34:224–8. 16. Vermorken JB, Remenar E, van Herpen C, et al. Cisplatin, fluorouracil, and docetaxel in unresectable head and neck cancer. N Engl J Med. 2007;357:1695–705. 17. Posner MR, Hershock DM, Biajman CR, et al. Cisplatin and fluorouracil alone or with docetaxel in head and neck cancer. N Engl J Med. 2007;357:1705–15. 18. Pointreau Y, Garaud P, Chapet S, et al. Randomized trial of induction chemotherapy with cisplatin and 5-fluorouracil with or without docetaxel for larynx preservation. J Natl Cancer Inst. 2009; 101:498–506. 19. Pignon JP, le Maitre A, Maillard E, et al. Meta-analysis of chemotherapy in head and neck cancer (MACH–NC): an update on 93 randomised trials and 17, 346 patients. Radiother Oncol. 2009; 92:4–14.

J.-L. Lefebvre 20. Merlano M, Vitale V, Rosso R, et  al. Treatment of advanced squamous-cell carcinoma of the head and neck with alternating chemotherapy and radiotherapy. N Engl J Med. 1992;327:1115–21. 21. Merlano M. Alternating chemotherapy and radiotherapy in locally advanced head and neck cancer: an alternative? Oncologist. 2006;11:146–51. 22. Forastiere AA, Goepfert H, Maor M, et al. Concurrent chemotherapy and radiotherapy for organ preservation in advanced laryngeal cancer. N Engl J Med. 2003;349:2091–8. 23. Forastiere AA, Maor M, Weber RS, et al. Long-term results of Intergroup RTOG 91-11: a phase III trial to preserve the larynxinduction cisplatin/5-FU and radiation therapy versus concurrent cisplatin and radiation therapy versus radiation therapy. J Clin Oncol. 2006 ASCO Annual Meeting Proc.2006;24:abstract 5517. 24. Machtay M, Moughan J, Trotti A, et  al. Factors associated with severe late toxicity after concurrent chemoradiation for locally advanced head and neck cancer: a RTOG analysis. J Clin Oncol. 2008;26:3582–9. 25. Lefebvre JL, Rolland F, Tesselaar M, et al. Phase 3 randomized trial on larynx preservation comparing sequential vs alternating chemotherapy and radiotherapy. J Natl Cancer Inst. 2009;101:142–52. 26. Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and Neck. N Engl J Med. 2006;354:567–78. 27. Posner MR, Norris CM, Wirth LJ, et  al. Sequential therapy for locally advanced larynx and hypopharynx cancer subgroup in TAX 324: survival, surgery, and organ preservation. Ann Oncol. 2009;20:921–7. 28. Worden FP, Moyer J, Lee JS, et al. Chemoselection as a strategy for organ preservation in patients with T4 laryngeal squamous cell carcinoma with cartilage invasion. Laryngoscope. 2009;119:1510–7. 29. Lefebvre JL, Pointreay Y, Rolland F, et al. Sequential chemoradiotherapy (SCRT) for larynx preservation (LP): preliminary results of the randomized phase II TREMPLIN study. PASCO Annual Meeting Proc. 2009:abstract 6010. 30. Lefebvre JL, Ang KK. Larynx preservation clinical trial design: key issues and recommendations – a consensus panel summary. Int J Radiat Oncol Biol Phys. 2009;73:1293–303. 31. Lefebvre JL, Ang KK. Larynx preservation clinical trial design: key issues and recommendations – a consensus panel summary. Head Neck. 2009;31:429–41.

Chapter 24

Multidisciplinary Approach of Unresectable Head and Neck Cancer Ricardo Hitt, Blanca Homet, and Robert I. Haddad

Abstract  The management of locally advanced head and neck cancer has seen the emergence of different combined modality therapies in recent years, and new treatment types, such as chemoradiation, new induction chemotherapy schemes, and salvage surgery.

better defined. Herein, we present some guidelines with respect to unresectability criteria, though these criteria may vary depending on the specialist.

Keywords  Unresectable head and neck cancer • Induction chemotherapy • Chemoradiation • Taxanes

Unresectable Disease: Definition

Introduction

The American Joint Commission on Cancer (AJCC) has recently revised its TNM classification to clearly separate T4 disease into two categories: Resectable or T4a and unresectable or T4b. The following is the official definition of unresectable or T4b disease per AJCC.

The management of locally advanced head and neck cancer (LAHNC) has seen the emergence of different combined modality therapies in recent years. These include surgery, chemoradiotherapy (CRT), induction chemotherapy (IC), sequential chemoradiotherapy (ST), and biologic therapy [1]. The management of this complex disease requires a multidisciplinary team including medical and radiation oncologists, head and neck and oral surgeons, speech and swallow therapists, psychiatrists, and dentists. The multi-D clinic role is valuable in determining the optimal staging, treatment and potential for rehabilitation after therapy. One of the more important factors in LAHNC patients is between resectable and unresectable disease. This is due to the differences in survival rates, locoregional control, and treatment in the two groups. Recently, this distinction has been taken into account in the design of clinical trials. It is clear based on randomized phase III studies that patients with unresectable disease have a worse prognosis with a high rate of both local and distant recurrence [2, 3]. There is no “consensus” on how to define unresectable disease. To improve the therapeutic approach in patients with nonsurgical disease, the term “Unresectable” will need to be

R.I. Haddad (*) Department of Medical Oncology, Dana–Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA e-mail: [email protected]

1. Oral cavity: Tumor invades masticator space, pterygoid plates, or skull base and/or encases internal carotid artery. The lesion must be so extensive that a functional reconstruction is not possible. 2. Oropharynx: Invasion of the lateral pterygoid muscle, pterygoid plates, lateral nasopharynx, or skull base or encases carotid artery. 3. Hypopharynx: Tumor invades prevertebral fascia, encases carotid artery, or involves mediastinal structures. 4. Larynx: Tumor invades prevertebral space, encases carotid artery, or involves mediastinal structures. The following is also important in further establishing resectable or unresectable disease: 1. For vascular encasement, involvement of 270° or more of the circumference of the carotid artery is accurate in predicting the surgeons’ inability to peel the tumor off the carotid artery in 100% of the cases [4, 5]. This criterion is often used to determine whether a tumor is unresectable. MR is the preferred imaging modality. 2. Involvement of the prevertebral fascia means the fixation of the tumor to the prevertebral musculature. The presence of a high-signal-intensity fat stripe on sagittal or axial T1-weighted scans by MRI shows the absence of infiltration of the prevertebral musculature with an accuracy of 91% [6].

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_24, © Springer Science+Business Media, LLC 2011

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3. Mediastinal invasion, which is more typical in infrahyoid tumors, is the infiltration of the mediastinal fat, vascular invasion of the supra-aortic vessels, or infiltration of the trachea and esophagus [5]. It is important to note that patient inoperability is often determined using three criteria: 1. Technical unresectability as previously detailed for T4b disease. 2. Low surgical curability such as seen in many patients with T4a disease and large and fixed neck adenopathy. 3. Medical contraindication to surgery.

Concurrent Chemoradiotherapy The use of concurrent chemoradiotherapy is considered by many to represent a standard of care in the management of patients with locally advanced and unresectable disease (Table 24.1). Studies have shown that combining chemotherapy with radiation improves local control and survival. Bolus cisplatin at 100 mg/m2 every 3 weeks is the drug of choice for patients with a good performance status [2]. The American Head and Neck Intergroup conducted a phase III randomized trial to study the benefit of adding chemotherapy to radiation in patients with unresectable squamous cell carcinoma of the head and neck [2]. Patients were randomly assigned between arm A, single daily fractionated radiation (70 Gy at 2 Gy/day); arm B, identical radiation therapy with concurrent bolus cisplatin at 100 mg/m2, given on days 1, 22, and 43; and arm C, a split course of single daily fractionated radiation and three cycles of concurrent infusional FU and bolus cisplatin chemotherapy, 30 Gy given with the first cycle and 30–40 Gy given with the third cycle. Surgical resection was encouraged if possible after the second chemotherapy cycle on arm C and, if necessary, as salvage therapy on all three treatment arms. The extent of midcourse surgery for arm C patients was defined on the basis of the residual disease present, not the original tumor. Two hundred and ninety-five patients were entered on this trial. Median age is 56 and half the patients had oropharynx primary. With a median follow-up of 41 months, the 3-year

overall survival for patients enrolled in arm A is 23%, compared with 37% for arm B (p = 0.014) and 27% for arm C (p = not significant). Chemotherapy did not affect the likelihood of distant recurrence when compared with radiation therapy alone. Distant metastases were the first site of recurrence in 17.9% of arm A patients, 21.8% of arm B patients, and 19.1% of arm C patients; the differences were statistically insignificant. When surgical results were analyzed, little difference in the rate of surgical resection was observed among the three treatment arms. Ultimately, 21% of all patients underwent surgery; neck dissection alone was performed in 56% of the surgical cases. Grade 3 or worse toxicity occurred in 52% of patients enrolled in arm A, compared with 89% enrolled in arm B (p < 0.0001) and 77% enrolled in arm C (p < 0.001). Major toxicities encountered were mucositis and feeding tube dependency and toxicity was worse in arm B. The authors concluded that the addition of concurrent high-dose, single-agent cisplatin to conventional single daily fractionated radiation significantly improves survival, although it also increases toxicity. Weekly low dose cisplatin has been tried and it does not appear to be as beneficial as high dose cisplatin with one study showing it to be equivalent to XRT alone [7]. Given the toxicity of high dose cisplatin, other regimens have been explored. Weekly carboplatin and paclitaxel is another regimen that can be used for those patients who cannot tolerate high dose cisplatin and phase II data suggest that the treatment can be effective. This regimen was explored in a single institution study with 55 patients [8]. Fifty-two patients (95%) had stage IV and 51 (93%) had technically unresectable disease; 62% had an oropharyngeal primary site. Grade 3 or 4 mucositis occurred in 30% of patients. Forty of 50 assessable patients (80%) had an objective response, with a complete response rate of 52%. With a median follow-up of 69 months for surviving patients, the 5-year progression-free survival was 36% and the 5-year overall survival was 35%. Another study from the University of Maryland group explored the same regimen of carboplatin/paclitaxel and standard daily radiation [9]. Sixty-two patients were treated with 70.2 Gy of RT at 1.8 Gy/fraction/ day to the primary site. Weekly chemotherapy was given during RT consisting of paclitaxel (45  mg/m2/week) and carboplatin (100  mg/m2/week). All patients presented with

Table 24.1  Summary of clinical trials in unresectable head and neck cancer Type Number Treatment Intergroup study [2]

Phase III

Agarwala et al. [8] Phase II University of Maryland [9] Phase II Medina et al. [10] Phase II ECOG [12] Phase II RT radiotherapy, CRT chemoradiotherapy

295

RT/CRT/split

  55   62   94   60

CRT CRT CRT CRT

Chemotherapy used

OS (%)

Bolus cisplatin (With 5-FU for split regimen) Weekly carboplatin and paclitaxel Weekly carboplatin/paclitaxel Weekly cisplatin Cisplatin q 3 weeks and cetuximab weekly

23/37/27 35 48 41 67

24  Multidisciplinary Approach of Unresectable Head and Neck Cancer

locally advanced disease; 77% had T4 disease and 21% had T3 disease. Fifty-eight percent had N2b–N3 disease. Ninetyeight percent of patients completed prescribed therapy. A clinical complete response at the primary site was obtained in 82%, with a total (primary site and neck) complete response rate of 75%. The median survival for the entire cohort is 33 months. Response to therapy and status of the neck at presentation were the only prognostic factors found to influence survival. The median survival for patients who attained a CR is 49 months versus 9 months in those who did not attain a CR. The 2- and 3-year overall survival for complete responders is 79 and 61%. The regimen was well tolerated with over 90% of patients completing prescribed therapy. With 48% 3-year overall survival for the entire group, this regimen is an acceptable choice for this group of patients with a historically poor prognosis. Given the poor outcome encountered in patients with unresectable disease other radiation modalities have been tried. Concomitant boost radiation which applies a second daily radiation treatment can result in decreased tumor repopulation, a major factor in local regional failure. A phase II study exploring weekly cisplatin with concomitant boost radiation has been recently reported [10]. In this study, a total of 94 patients (median age, 58 years) with cancers of the oropharynx, larynx, hypopharynx, and oral cavity were included. Patients received radiotherapy with a concomitant boost scheme (1.8  Gy on days 1–40 and 1.5  Gy boost on days 25–40 with a total dose of 72 Gy) and concurrent cisplatin, 40  mg/m2 weekly, for the first 4 weeks only. Most patients (95%) received both radiation and chemotherapy according to protocol. Toxicity was manageable. With a median follow-up of 41 months, median overall survival and time to progression were 27 and 25 months, respectively. The estimated overall survival at 4 years was 41%. The poor results encountered with standard chemotherapy regimens have also prompted studies of novel agents. EGFR inhibitors appear to be the most promising class of drugs [11]. Recently, investigators from the Eastern Cooperative Oncology Group (ECOG) reported their first study of CRT with cetuximab in unresectable head and neck cancer [12]. In this study, patients with unresectable, newly diagnosed head and neck cancer received cetuximab, an EGFR inhibitor,

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400 mg/m2 on day 1, then 250 mg/m2 weekly, in combination with definitive radiation therapy (70 Gy/2 Gy/day × 7 weeks) starting day 15 and cisplatin 75 mg/m2 every 3 weeks. In the absence of disease progression or untoward toxicity, patients could continue cetuximab weekly for up to 1 year. In this trial, 60 patients were treated; median age was 56 and 98% were stage IV. Most common primary sites included base of tongue (34%), tonsil (21%), and other oropharynx (13%). One-year survival is 76% and projected 2-year survival is 67%. Median survival is 33 months. Unique toxicities include acneiform rash and an increase in severe mucositis. These early results are promising and do represent a significant improvement over cisplatin/radiation regimens. Further trials are underway to further study this regimen.

Sequential Treatment As mentioned above, with CRT or radiation treatment alone, locoregional control (LRC) and survival rates in patients with unresectable LAHNC is quite poor. The use of new induction chemotherapy regimens with taxanes added to platinum–5-FU (PF) results in a high response rate and better survival compared to the traditional PF schedule. Taxanebased chemotherapy was not analyzed in the MACH metaanalyses, where patients included received different modality treatments with induction chemotherapy and CRT, and stratification according to resectable or unresectable tumors did not take place [13]. There are many studies that have examined the addition of taxanes to PF and these combinations do represent a significant improvement over PF in term of efficacy and toxicity (Table 24.2). The safety and efficacy of docetaxel with PF (TPF) as induction chemotherapy for patients with SCCHN were evaluated in a multicenter, open-label, randomized trial (Tax 323) [3]. In this European study, 358 patients with SCCHN with previously untreated inoperable, locally advanced stages III and IV, and good performance status, received either docetaxel 75 mg/m2 followed by cisplatin 75 mg/m2 on day 1, followed by 5-FU 750  mg/m2/day as a continuous intravenous infusion on days 1–5 (TPF), or cisplatin 100 mg/m2 on

Table 24.2  Summary of phase III induction chemotherapy trials in unresectable head and neck cancer Study Regimen Number Endpoint TAX 324 [14] TAX 323 [3] Spanish Intergoup [16]

TPF vs. PF followed CRT TPF vs. PF followed CRT or RT PF or TPF followed CRT vs. CRT

501 358 439

Overall survival (months) Progression-free survival (months) Time to treatment failure (months)

Results

71 vs. 30 (p = 0.006) 11.0 vs. 8.2 (p = 0.007) Induction chemotherapy plus CRT = 12.5 (median) vs. CRT = 5 (median) p = 0.0001 Spanish Intergroup [15] PCF vs. PF followed CRT 382 Complete response (CR) 33 vs. 14% (p = 0.001) TPF docetaxel, cisplatin, and 5-FU, PF cisplatin and 5-FU, PCF paclitaxel, cisplatin, and 5-FU, CRT chemoradiotherapy

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day 1, followed by 5-FU 1,000 mg/m2/day as a continuous intravenous infusion on days 1–5 (PF). These regimens were administered every 3 weeks for four cycles. Four to 7 weeks after chemotherapy, patients whose disease had not progressed received radiotherapy. Radiation was delivered either with a conventional or an accelerated/hyperfractionated regimen (i.e., more than one fraction per day). Surgical resection was allowed following chemotherapy, before or after radiotherapy. The trial’s primary endpoint was progression-freesurvival (PFS) that was defined as time from randomization to disease progression or death from any cause, whichever occurred first. Median PFS was significantly longer in the TPF arm (11.4 months) than in the PF arm (8.3 months). Median overall survival was significantly longer in the TPF arm (18.6 months) than in the PF arm (14.2 months). The FDA approved this regimen for patients with inoperable SCCHN on October 17, 2006. Tax 324 [14] took a different approach from TAX 323. Patients included in this study had both operable and inoperable disease. The clinical observations of the last three decades that concurrent chemoradiotherapy is crucial in SCCHN were taken into account when this study was designed, and instead of giving radiation therapy only after IC, all patients received concurrent chemoradiotherapy. The goal is to combine both models of therapy in one study: IC and concurrent chemoradiotherapy. This is an international multicenter, open-label, randomized phase III trial. In this study, 501 patients with previously untreated locally advanced SCCHN, and good performance status, received either docetaxel 75 mg/m2 followed by cisplatin 100 mg/m2 on day 1, followed by 5-FU 1,000  mg/m2/day as a continuous intravenous infusion on days 1–4 (TPF) or cisplatin 100 mg/m2 on day 1, followed by 5-FU 1,000 mg/m2/day as a continuous infusion on days 1–5 (PF). These regimens were administered every 3 weeks for three cycles. All patients in both treatment arms who did not have progressive disease following induction chemotherapy (close to 80%) received 7 weeks of CRT. During radiotherapy, carboplatin, area under the curve (AUC) of 1.5 was administered weekly as a 1-h infusion for a maximum of seven doses. Surgery could be considered at anytime following the completion of CRT. The majority of patients had locally advanced stage IV disease (84%). Overall survival was significantly prolonged with TPF compared to PF regimen (log-rank test, p = 0.0058). The median survival was 70.6 months in the TPF group compared to 30.1 months in the PF group. There is an increase in the incidence of neutropenia and febrile neutropenia with TPF and more stomatitis/diarrhea with PF likely reflecting the higher dose of 5-FU dose used. In TAX 324, close to 70% of patients completed the concurrent chemoradiotherapy regimen as defined per protocol. The major reasons for not completing CRT are disease

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progression and adverse events. Two treatment-related deaths related to induction chemotherapy occurred in TAX 324. In TAX 323, close to 70% of patients completed radiotherapy per protocol, with disease progression as the main reason for not completing the protocol. Five treatment-related deaths related to induction chemotherapy occurred in this study. The Spanish group examined the addition of Paclitaxel to PF in a randomized phase III study [15]. The primary objective is to compare the activity and toxicity of the two induction chemotherapy treatments of paclitaxel, cisplatin, and FU (PPF) versus standard cisplatin and FU (PF), both followed by CRT, in locally advanced and unresectable head and neck cancer. Both regimens were administered for three cycles every 21 days. Patients with complete response (CR) or partial response of greater than 80% in primary tumor received additional CRT (cisplatin 100 mg/m2 on days 1, 22, and 43 plus 70 Gy). A total of 382 eligible patients were randomly assigned to PF (n = 193) or PPF (n = 189). The CR rate was 14% in the PF arm versus 33% in the PPF arm (p < 0.001). Median time to treatment failure (TTF) was 12 months in the PF arm compared with 20 months in the PPF arm (p = 0.006). PPF patients had a trend to longer overall survival that was not statistically significant. This difference was more evident in patients with unresectable disease. PF patients had a higher occurrence of grade 2–4 mucositis than PPF patients (53 vs. 16%, respectively; p < 0.001). However, the induction chemotherapy plus CRT approach was limited to a select group of patients because of the significant toxicity produced by such treatment. Six cycles of cisplatin (induction plus chemoradiation) is possible only in patients with excellent performance status, adequate organ function, and intensive medical support. This significantly limits the use of this regimen. Finally, the same Spanish group recently presented the data of a randomized phase III trial [16], where induction chemotherapy (With TPF or PF) plus CRT was compared with standard CRT as front line treatment in patients with unresectable locally advanced head and neck cancer. The primary end point of this study was TTF for induction versus no induction chemotherapy; secondary endpoints included LRC. In evaluable patients, the median TTF was 12.5 months with induction plus CRT versus 4.9 months with CRT alone (p < 0.001). LRC was 60% with induction chemotherapy plus CRT versus 44.5% with CRT alone (p = 0.003). Further analysis of this trial is ongoing and the results should be viewed as preliminary at this point.

Discussion The management of patients with LAHNC requires a multidisciplinary evaluation. Differentiation between resectable and unresectable disease is important and carries significant

24  Multidisciplinary Approach of Unresectable Head and Neck Cancer

prognostic implications. Patients with unresectable head and neck cancer have a worse prognosis and novel therapies are needed for this patient population. A multidisciplinary approach for these patients is crucial and helps with staging, treatment decision, and management of the acute and longterm complication of therapy. A better definition for unresectable disease is needed. Currently, there are two acceptable standards for these patients: concurrent chemoradiotherapy and induction chemotherapy followed by CRT. Each approach has its own advantages and disadvantages. We would recommend induction chemotherapy for the following patients: 1. Symptomatic patients in need for immediate therapy or patients with impending local problems such as locally advanced larynx cancer where airway compromise is imminent. 2. Patients with high risk of distant metastasis such as nodal disease N2b, N2c, and N3 patients. 3. Patients with possible or proven distant metastasis at presentation. For these patients, induction chemotherapy with TPF is our choice for therapy followed by concurrent chemoradiotherapy with weekly carboplatin and radiation based on the results of TAX 324. All other patients with unresectable disease can be treated with concurrent chemoradiotherapy upfront with either bolus cisplatin every 3 weeks or weekly carboplatin/paclitaxel. Given the overall poor outcome for these patients, novel approaches are urgently needed. Two such approaches have shown early promise: accelerated radiotherapy and the addition of novel targeted agents. Early results appear to show some improvement over standard therapy and further studies are ongoing to define the optimal strategy for these patients.

References 1. Haddad RI, Shin DM. Recent advances in head and neck cancer. N Engl J Med. 2008;359:1143–54. 2. Adelstein DJ, Li Y, Adams GL, Wagner Jr H, Kish JA, Ensley JF, et al. An intergroup phase III comparison of standard radiation therapy and two schedules of concurrent chemoradiotherapy in patients with unresectable squamous cell head and neck cancer. J Clin Oncol. 2003;21:92–8.

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3. Vermorken JB, Remenar E, van Herpen C, Gorlia T, Mesia R, Degardin M, et al. Cisplatin, fluorouracil, and docetaxel in unresectable head and neck cancer. N Engl J Med. 2007;357:1695–704. 4. Yousem DM, Hatabu H, Hurst RW, Seigerman HM, Montone KT, Weinstein GS, et al. Carotid artery invasion by head and neck masses: prediction with MR imaging. Radiology. 1995;195:715–20. 5. Yousem DM, Gad K, Tufano RP. Resectability issues with head and neck cancer. AJNR Am J Neuroradiol. 2006;27:2024–36. 6. Hsu WC, Loevner LA, Karpati R, Ahmed T, Mong A, Battineni ML, et al. Accuracy of magnetic resonance imaging in predicting absence of fixation of head and neck cancer to the prevertebral space. Head Neck. 2005;27:95–100. 7. Haselow R, Warshaw M, Oken M, et  al. Radiation alone versus radiation with weekly low dose cis-platinum in unresectable cancer of the head and neck. In: Fee Jr WE, Goepfert H, Johns ME, et al., editors. Head and neck cancer, vol. II. Philadelphia, PA: Lippincott; 1990. p. 279–81. 8. Agarwala SS, Cano E, Heron DE, Johnson J, Myers E, Sandulache V, et  al. Long-term outcomes with concurrent carboplatin, paclitaxel and radiation therapy for locally advanced, inoperable head and neck cancer. Ann Oncol. 2007;18:1224–9. 9. Sutharalingum M, Haas M, Conley B, Egorin M, Levy S, Sivisailam S, et al. The use of carboplatinum and paclitaxel with daily radiotherapy in patients with locally advanced squamous cell carcinomas of the head and neck. Int J Radiat Biol Oncol Phys. 2000;47:49–56. 10. Medina JA, Rueda A, de Pasos AS, Contreras J, Cobo M, Moreno P, et al. A phase II study of concomitant boost radiation plus concurrent weekly cisplatin for locally advanced unresectable head and neck carcinomas. Radiother Oncol. 2006;79:34–8. 11. Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 2006;354:567–78. 12. Langer C, Lee J, Patel U, Shin D, Argiris A, Quon H et  al. Preliminary analysis of ECOG 3303: concurrent radiation (RT), cisplatin (DDP) and cetuximab (C) in unresectable, locally advanced (LA) squamous cell carcinoma of the head and neck (SCCHN). J Clin Oncol 2008;26:(May 20 suppl; abstr 6006). 13. Pignon JP, Bourhis J, Domenge C, Designe L. Chemotherapy added to locoregional treatment for head and neck squamous-cell carcinoma: three meta-analyses of updated individual data. MACH-NC Collaborative Group. Meta-Analysis of Chemotherapy on Head and Neck Cancer. Lancet. 2000;355:949–55. 14. Posner MR, Hershock DM, Blajman CR, Mickiewicz E, Winquist E, Gorbounova V, et  al. Cisplatin and fluorouracil alone or with docetaxel in head and neck cancer. N Engl J Med. 2007;357:1705–15. 15. Hitt R, Lopez-Pousa A, Martinez-Trufero J, Escrig V, Carles J, Rizo A, et  al. Phase III study comparing cisplatin plus fluorouracil to paclitaxel, cisplatin, and fluorouracil induction chemotherapy followed by chemoradiotherapy in locally advanced head and neck cancer. J Clin Oncol. 2005;23:8636–45. 16. Hitt R, Grau J, Lopez-Pousa A, Berrocal A, Garcia Giron C, Irigoyen A et al. Final results of a randomized phase III trial comparing induction chemotherapy with cisplatin/5-FU or docetaxel/cisplatin/5-FU followed by chemoradiotherapy (CRT) versus CRT alone as first-line treatment of unresectable locally advanced head and neck cancer (LAHNC). J Clin Oncol 2009;27:15S, (suppl; abstr 6009).

Chapter 25

Laser Endoscopic Treatment Pierre R. Moreau and Pierre H. Demez

Abstract  After a review of CO2 laser technique in the treatment of head and neck cancers, the results of the literature are presented for each localization. For early glottic cancers T1–T2, the specific survival rate at 5  years is around 100%, with a local recurrence rate of 10% and an incidence of total laryngectomy limited to 2–3%, lower than after radiotherapy. For supraglottic cancers, the expertise is important; techniques, indications, and results differ depending on the authors. One observes 10% of local recurrence for T1–T2 and 20% for T3–T4, with a specific survival rate of 80% at 5 years. This disparity is stronger for pharyngeal cancers. For precancerous lesions, laser gives a local recurrence rate around 10%, which can be salvaged without total laryngectomy –contrary to postradiation salvage –with a specific survival rate near 100%. Laser debulking of obstructing tumors can be performed in order to avoid tracheotomy. Postradiation recurrence can be salvaged by laser only for a few parts, with an important rate of new recurrences and total laryngectomies. Keywords  CO2 laser • Endoscopic surgery • Transoral laser surgery • Glottic • Supraglottic • Pharyngeal carcinoma • Laryngeal carcinoma in situ • Glottic dysplasia • Airway obstruction • Recurrent laryngeal carcinoma

Introduction Endoscopic removal of early laryngeal cancers was reported as early as 1915 [1]. The development of direct suspension laryngoscopy and subsequent use of microscopic examination was pioneered by Chevalier Jackson in the 1930s and Oskar Kleinsasser in the 1960s. The electric bistoury was introduced to surgery by Cushing in 1926, following the work of Bovie [2]. Einstein developed the theoretical design of the laser in 1917. The first pulsed ruby laser was described by Maiman

P.R. Moreau (*) Department of Otorhinolaryngology, University Hospital of Liege, Liege, Belgium e-mail: [email protected]

in 1960, then used a few months later to treat a retinal tumor [3]. The carbon dioxide laser (CO2 laser), coupled with use of the microscope in direct suspension laryngoscopy, was first used by Jako and Strong in the early 1970s, and subsequently by other members of the Boston University group [4]. Used principally for benign lesions, in 1975, Strong reported three cases of laryngeal cancer excision using the CO2 laser [5]. In the early 1980s, Wolfgang Steiner was responsible for the development and growth of CO2 laser in the treatment of head and neck cancers. Table 25.1 presents an historical synopsis. The term “laser” is an acronym for “light amplification by stimulated emission of radiation.” It consists of a spatially and temporally coherent beam of light produced by amplifying a stimulated emission beam, enabling a large amount of energy to be concentrated upon a small surface. Following production of the first ruby laser in 1960, various types of medical lasers were developed, differing in terms of their physical characteristics. The argon laser has coagulative properties, the Nd:YAG laser has absorptive properties, and the CO2 laser has cutting properties. Because the CO2 laser beam is invisible, a ­red-­colored coaxial helium–neon beam is used to enable localization. A micromanipulator mounted on a mirror allows maneuvering of the beam. The length of a CO2 laser wave results in it having a high capacity to absorb water, and thus tissue, resulting in heating and destruction of tissue. The first CO2 lasers had a spot of approximately 1  mm in size. As a result of subsequent progress, the size of the spot has been reduced to around 200 mm, for example with the Acuspot. In contrast to the electric bistoury, which is active when in contact with tissue, the laser is used at a distance, allowing it to be used on the larynx and hypopharynx. The CO2 laser is the principle laser used in the treatment of head and neck cancers, and this chapter will be devoted to discussions of this tool.

Laser Techniques Precautions are required when using a CO2 laser, as it is capable of being reflected and the resulting heat is liable to ignite. All operating theater staff must wear eye protection.

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_25, © Springer Science+Business Media, LLC 2011

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340 Table 25.1  Historical synopsis 1915 Lynch [1] Endoscopic resection of nine early glottic cancers 1917 Einstein Theoretical design of laser 1960 Maiman [3] Ruby laser for retinal tumor 1970 Jako-Strong [4] CO2 laser for benign laryngeal lesions 1975 Jako-Strong [5] CO2 laser for three laryngeal cancers 1980 Steiner [6] CO2 laser for head and neck cancers 1992 Eckel [7] 67 T1–T2 glottic cancers 1993 Steiner [8] 130 T1–T2a glottic cancers 1998 Iro [9] 141 supraglottic cancers 2001 Steiner [10] 129 pyriform sinus cancers 2005 Motta [11] 719 T1–T3 glottic cancers 2008 Martin [12] 172 hypopharyngeal cancers

The patient’s eyes are closed and a cloth covers the patient’s face around the laryngoscope. The endotracheal ventilation tube should be a Mallinckrodt, Xomed, or other specific laser tube designed such that it will not catch fire when touched by the laser beam. A moist cotton pad must protect the inflated cuff. Another ventilation solution consists of using a supraor subglottic ventilation jet, which has the inconvenience of moving the laryngeal structures with each insufflation. Whether a ventilation tube or ventilation jet is used, it is recommended that the oxygen level be limited to 30% of the ventilation gas. Various methods and techniques facilitate the use of the laser. As with diagnosis using a direct microlaryngoscopy, the use of a head clamp, a remote-controlled operating table, a height-adjustable chair, direct and lateral viewing optics via the laryngoscope, and palpation by microforceps are required. A direct view of the anterior part of the larynx is always more difficult to attain. A tooth guard, possibly made of a thermoplastic material for making Kerr type dental molds, is useful. Manual pressure by a nurse on the patient’s neck is guided by a screen, allowing direction of the pressure to improve visibility. The loose movement of the epiglottis within the larynx can distort the anterior view. This can be remedied by passing a stitch through the epiglottis, holding it to the side of the laryngoscope while it is reinserted. A large suction tube is fixed to the laryngoscope to extract smoke produced by tissue vaporization. The laser beam is always used at the highest magnification possible while cutting, in order to destroy the least amount of tissue possible and allow for greater reliability of histological margins. The Acuspot allows the width of the section line to be reduced to, at best, 200  mm. The laser beam coagulates small vessels, while bleeding from larger vessels is stemmed using a monopolar suction-coagulator. The resected specimen is always spread out and oriented upon a support such as a corkboard, in such a way as to enable precise histological analysis of the margins. So, adequate laser re-resections are feasible in case of involvement. After removal of the specimen, tissue remaining at the level

P.R. Moreau and P.H. Demez

of the vocal cord is adjusted to improve voice result. In the event that a large amount of cartilage is exposed, it is standard procedure to prescribe antibiotic therapy to prevent chondritis. Postintervention scarring often lasts 2–3 months or more; granulomas may form and can be mistaken for an early recurrence. Some surgeons recommend a second look under general anaesthesia at 2 or 3 months postoperatively, and some even perform a third look [13].

Glottic Cancers Before the creation of CO2 lasers, early laryngeal cancers were treated endoscopically. In 1915, Lynch reported nine cases of endoscopic resection [1]. In 1973, Lillie and De Santo obtained excellent results in a series of 57 patients [14]. Nevertheless, it wasn’t until the 1972 description by Strong and Jako of the use of the CO2 laser, coupled with direct suspension microlaryngoscopy, that endoscopic resection really gained popularity [4]. The first publications reported between 1985 and 1990 included a limited number of patients and had only a short oncologic follow-up. Following Steiner, German teams were the first to publish a series of 100 patients with survival rate calculation, between 1990 and 1995 (Eckel and Thumfart [7], Steiner [8], Rudert and Werner [15]). Subsequently, other teams published their oncologic and functional results, although points of controversy still remained (see Table 25.1). In the English-speaking world, radiation coupled with salvage surgery is a common approach in the treatment of early glottic cancers. Radiation is accompanied by a high rate of recurrence, approximately 10% for T1 and 30% for T2 tumors, according to various authors and publications [16]. Partial open surgery results in a recurrence rate of around 5% [17, 18]. The use of laser technique and its indications are controversial. Minimalists limit its use to small cancers of the medial third of the vocal cord while maximalists treat even advanced T4 laryngeal cancers, involving endoscopic resection of cartilaginous segments. Depending upon the indications, the technique and the results are highly variable. For small tumors, the majority of surgeons advise en bloc resection. For larger tumors, some surgeons apply the same principle while others, following Steiner, recommend piecemeal resection, using Moh’s technique [6]. The European Laryngological Society’s classification system is widely used in Europe [19]. Cordectomy is classified as superficial, transmuscular, or radical, possibly extending to the anterior commissure, the arytenoid, the ventricular fold, or subglottis [20]. Anterior commissure extension is the subject of much debate, due to the possible risk of tumoral extension along the Broyle ligament. For some surgeons, even a superficial

25  Laser Endoscopic Treatment

extension of the anterior commissure is a contraindication against endoscopy. For many others, it is not a contraindication as long as the extension is superficial. And for yet other surgeons, even significant extension does not constitute a contraindication [21, 22]. A superficial supraglottic or subglottic extension does not constitute a real contraindication for many authors. Decreased mobility (T2b) requires care and necessitates a radical cordectomy with removal of the entire muscular thickness [23]. Extension of the contralateral vocal cord leads to a synechia and strongly alters the vocal result, rendering this procedure controversial. Finally, only a few authors advise piecemeal cartilagineous excision for T3 or T4 lesions [8, 24]. Most authors attach crucial significance to the histological examination of superficial and deep margins, but this is not always the case. Some recommend a second examination a month or two after initial surgery to confirm that there has been no recurrence [13]. We do not see the need for this if the histological specimen removed en bloc has been analysed with care and shows no tumor at the surgical margins. Our only indication for revision under general anaesthesia is that of a granuloma that lasts more than 4 or 5 months or which alters vocal quality. Large series with sufficient oncologic follow-up are now available. Table 25.2 summarises the most significant publications. Several series include 200–300 patients, with Motta publishing results for 400 cases. For T1–T2 and excluding T3 lesions, the adjusted survival rate at 5  years is close to 100%. Patients do not die as a result of their glottic tumor. Motta alone reported less favorable results. The local recurrence rate is around 10%, varying from 0% to 20%. Treatment of these recurrences is effective. A total laryngectomy rate of approximately 2–3% is reported, ranging from 0% to 10%, and higher in the case of T2 and T3 tumors. The overall survival rate at 5 years is around 80%. Numerous studies

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demonstrate that failures are more frequent in the event of involvement of the anterior commissure, in the presence of decreased mobility, and even more so in the event of glottic fixation or when cartilage is affected [11, 22, 23]. Usually, laser treatment is not followed by radiation. Some surgeons recommend postoperative radiotherapy when compounding factors exist, such as involvement of the anterior commissure or decreased mobility [23]. In light of this literature, it has become clear that laser resection gives better results that radiation or partial open surgery for the majority of T1 and T2 glottic tumors. This superiority should be qualified in light of the significant expertise required to carry out laser resection. Less favorable results are reported in certain limited series [31]. Moreover, series with the worst outcomes are not even published. The problem of anterior commissure involvement and decreased mobility remain, with both laser resection and radiation therapy producing less favorable results. As far as we are concerned, laser resection does not rule out the possibility of partial open surgery in T1 and T2 glottic cancers found to have significant anterior commissure involvement, or found to be immovable with microinstruments. Few authors advise endoscopic treatment of T3 and T4 glottic cancers [8, 24]. When we consider the treatment of glottic cancers, we should also consider the resulting vocal quality. Studies attempt to compare three treatment methods: radiotherapy, laser surgery, and open surgery [27]. The occurrence of salvage total laryngectomies is higher after radiation than after laser resection (see Table 25.2), reducing the quality of vocal results obtained by radiotherapy. When laser resection is extended to the contralateral cord, synechia alter vocal ­quality. Radical cordectomy up to the cricoid only results in a compensatory voice being produced via the supraglottis.

Table 25.2  Literature review concerning laser of glottic carcinomas Number Local Authors Year of patients Classification recurrence % Eckel [7] 1992 T1–T2 67 9 Steiner [8] 1993 130 T1–T2a 8 Rudert [15] 1995 108 T1–T2 9 Eckel [25] 2000 285 Tis–T2 14 Moreau [20] 2000 97 T1–T2 0 Gallo [26] 2002 139 T1 6 Brøndbo [27] 2004 118 T1a 10 Mortuaire [28] 2004 110 Tis–T1–T2 20 Peretti [29] 2004 322 Tis–T1–T2 9 Steiner [21] 2004 263 T1–T2a 13 Motta [11] 2005 432 T1 15 236 T2 34 51 T3 37 Peretti [23] 2005 55 T2 23 Ledda [30] 2006 103 Tis–T1–T2  3

Salvage total laryngectomy %

5-year specific survival %

5-year overall survival %

9 1 3 6 0 0 2 8 3 3 3 18 20 15  0

100 100 100 99 97 100 99 97 99 – 97 87 72 100 –

– 86 – 71 78 – – 87 88 – 85 77 64 76 92

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Supraglottic Cancers

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Endoscopic resection of limited supraglottic cancers was reported by Jackson and Jackson in 1939 [32]. After Jako and Strong, in 1978 Vaughan was the first to describe the use of resection using a CO2 laser for neoplasms of the suprahyoid epiglottis [4, 33]. Following his example, Zeitels and Davis used the CO2 laser for small cancers and to remove obstruction of tumors causing dyspnea, routinely following the endoscopic operation with radiation treatment [34]. It was in Europe, with Steiner in 1979 followed by Rudert, Motta, and Eckel, that CO2 laser endoscopic resection of supraglottic cancers really developed, without the use of systematic postoperative radiation treatment [15, 35–37]. A rise in the number of glottic cancers in the 1990s allowed the oncologic efficacy of endoscopic resection using the CO2 laser to be demonstrated on several series of hundreds of patients. In contrast, supraglottic cancers are more rare and treatment indications are more controversial, hence the current literature includes primarily reports of only 30–40 patients with short oncologic follow-up. Currently, only a few authors have published series of a 100 or more patients. The surgeon-dependent nature of this type of exercise calls for care in interpreting results and does not enable generalizations to be made. Steiner was the real pioneer in developing the technique of piecemeal resection for the removal of large supraglottic

cancers, extending its indication even to T4. Others remain loyal to en bloc resection, with more limited indications for laser use. The use of the bivalve laryngoscope as well as thicker forceps and suction tubes is indispensable for this type of resection. For small, limited tumors classed as T1, which are rare, all authors recommend en bloc resection. As soon as tumors become larger, Steiner recommends his piecemeal approach. Resection is carried out craniocaudally and layer by layer, using Moh’s technique (Fig.  25.1a, b). The first lateral incision cuts across the tumor on the median sagittal plane, allowing the surgeon to evaluate tumoral depth and thus the amount of tissue requiring removal. If the tumor is bulky, additional sections are carried out across the tumor. Where required, the thyroid cartilage or anterior glottal commissure is resected, thus an endolaryngeal evisceration is carried out. As far as we are concerned, we remain convinced that en bloc resection enables greater certainty in the analysis of histological margins than piecemeal resection (Fig. 25.1c). Where the preepiglottic space is involved this is not a contraindication if the involvement is minor, that is to say, if it remains far from the hyoid bone. It may reach close to the thyroid cartilage without affecting it. Extension of the anterior commissure is, for us, a contraindication to CO2 laser resection. The classically quoted risk of lymph node involvement is around 30% for supraglottic tumors and higher for tumors of

Fig. 25.1  Piecemeal (a, b) or en bloc (c) supraglottic resection. (a, b) From Rudert HH, Werner JA, Höft S. Transoral carbon dioxide laser resection of supraglottic carcinoma. Ann Otol Rhinol Laryngol 1999;108:819–827. Reprinted with permission from John Wiley

& Sons. (c) From Moreau P. Treatment of laryngeal carcinomas by laser endoscopic microsurgery. Laryngoscope 2000;110:1000– 1006. Reprinted and modified with permission from John Wiley & Sons

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Table 25.3  Literature review concerning laser of supraglottic carcinomas Number Authors Year of patients Classification Eckel [7] 1992 Zeitels [34] 1994 Eckel [37] 1997 Ambrosch [35] 1998 Iro [9] 1998 Rudert [39] 1999 Moreau [20] 2000 Motta [36] 2004 Davis [38] 2004 Cabanillas [40] 2008 a Recurrence –free survival b 3-year survival c Non actuarial survival

15 19 46 48 141 34 18 124 46 26

T1–T2 T1–T2 T1–T2 T1–T2 T1–T4 T1–T4 Tis–T3 T1–T2–T3 T2 T1–T3

the aditus. The majority of authors recommend carrying out a bilateral neck dissection; unilateral dissection is appropriate when the tumor is highly lateralised. In the event of a very superficial microinvasive tumor, the indication for neck dissection remains controversial. Postoperative radiation is indicated in the event of lymph node involvement, particularly significant involvement, and in cases where tumor margins are not resectable endoscopically. For some surgeons, such as Davis, postoperative radiation is routine [38]. Table 25.3 presents the most significant published series. Among the 517 patients identified in this literature compilation, only two authors report a group of more than 100 patients. For T1 and T2 tumors, the local recurrence rate varies between 0% and 10%, with the exception of Motta. When T3 and T4 are included in the series, the local recurrence rate rises to 0–30%. The compilation found 76 recurrences in 517 patients, for a rate of 15%. A large part of these recurrences improved with effective salvage treatment. The adjusted survival rate at 5 years varies between 70% and 100%, dependent primarily upon lymph node involvement and distal metastases. The overall survival rate at 5 years is on the order of 60–70%. The unanimously recognised advantages of this approach include the avoidance of tracheotomy, simpler postoperative course than with open supraglottic laryngectomy, and more rapid removal of nasogastric tubes [41]. Salvage treatment after recurrence with an endoscopic resection is clearly more effective than after radiation or open surgery.

Pharyngeal Cancers While the CO2 laser is increasingly used for the treatment of glottic laryngeal cancers, its use to treat the pharynx remains completely marginal, and few publications exist.

Local recurrence % 0 0 9 8 16 29 0 18-33-23 3 8

5-year specific survival %

5-year overall survival %

72 83a 66a 80b 100 97-94-81

59 76

80

62b 63 82-59-51 63c –

The overall survival rate of cancers of the pharynx is not favorable, with a survival rate at 5 years of 50% for oropharyngeal cancers and of 30% for hypopharyngeal cancers. Wolfgang Steiner pioneered the use of CO2 laser to treat cancers in different regions of the head and neck, notably of the pharynx, at the beginning of the 1980s. He replaced the use of the electric bistoury with the CO2 laser for all transoral resections, whether in the oral cavity itself, the oropharynx, or the hypopharynx. Others, including us, have reserved the use of the CO2 laser to regions that are inaccessible for transoral resection using an electric bistoury. The material used for laser resection of pharyngeal lesions is comparable to that used for supraglottic cancers, requiring the use of a bivalve Weerda-type laryngoscope and thicker and more rigid forceps and suction tubes. The indications for laser resection vary widely depending upon the author. Some surgeons use this technique only in a minority of the pharyngeal cancers they treat, limiting its indication to small early tumors, which are resectable en bloc with healthy superficial and deep histological limits [42, 43]. Scanner data and mobilisation of the tumor with the help of microforceps allow the depth of the extension to be evaluated and more precise evaluation to determine the appropriateness of laser resection. In contrast, Steiner and others use it to treat T3 and T4 tumors. He collated 31 pT3-T4 cancers from 129 laser resections [10]. As soon as the tumor reaches more than 1  cm in diameter, he recommends sectioning through the tumor and removing it piecemeal craniocaudally and layer by layer, according to Moh’s technique (Fig. 25.2) [6]. All the authors agree that histological analysis of the tumor margins is crucial and requires the correct orientation of the specimen and a meticulous analysis of the superficial and deep margins, whether the resection is carried out en bloc or piecemeal [44, 45]. Steiner claims that transsectioning the tumor in this way enables him to better evaluate the

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Fig. 25.2  Piecemeal (a) or en bloc (b) pharyngeal resection

Table 25.4  Literature review concerning laser of pharyngeal carcinomas Number Local 5-year specific* or rec. free** survival % Authors Year of patients Localization recurrence % Steiner et al. [10] 2001 Pyriform sinus 13 129 76** Steiner et al. [46] 2003   48 Base of tongue 15 73** Rudert and Höft [42] 2003   29 Hypopharynx 28 58* Vilaseca et al. [47] 2004   28 Hypopharynx 18 59* Martin et al. [12] 2008 172 Hypopharynx 26 72* Moreau et al. [43] 2008   74 Oro-hypopharynx  9 91* N.B. The study of Steiner 2001 is included in Martin 2008. The survival of Vilaseca is at 4, and not 5-year *  5-year specific survival **  5-year recurrence free survival

depth of the infiltration and prevents hindrance by the tumoral volume. Others claim that histological analysis of all limits is more accurate with en bloc resection. Unilateral or bilateral neck dissection is carried out depending upon the location of the tumor, either during the same operation or a few weeks later. This delay is required when there is a risk of communication between the two fields. For Rudert, postoperative radiation is routine [42]. For others, it is routine only in the event of lymph node involvement [43]. For Steiner, it is systematic in the event of advanced lymph node involvement, extracapsular spread, or carcinomatous lymphangitis [12]. Table  25.4 presents the results of the largest published series. In terms of the oropharynx, only the Steiner series, with 48 cases of localisation at the level of the base of the tongue, and Moreau, with 38 cases of various oropharyngeal localisations, exist [43, 46]. For the hypopharynx, the 172 Martin cases including the 129 cases published by Steiner are the only significant series [10, 12]. The remaining publications are limited to around 30 patients. For early stage T1 and T2N0, Steiner, Martin, and Moreau find an adjusted survival rate at 5 years of 90–100%. For all stages, the adjusted survival rate at 5 years is around 70%, while the overall survival

5-year overall survival % 53 52 48 43 52 49

rate is around 50%. This difference is linked to second primaries, which are particularly common. The local recurrence rate ranges between 10% and 30%. Effective treatment of these recurrences is possible, whether by repeated laser resection, radiation therapy, or open surgery. One big advantage results from the simplicity of post­ operative courses, with a reduction in the length of hospitalization, the avoidance of tracheotomy, and recovery of swallowing function and phonation. Bleeding is an immediate postoperative complication in around 5% of cases, and can be fatal [12, 43, 47].

Precancerous Lesions In 1923, Jackson introduced the concept of pre-cancerous lesions of the larynx [48]. In 1952, Altman reported the first studies of in situ laryngeal carcinomas, analogous to those of the cervix [49]. In 1974, Strong described the use of the CO2 laser for the treatment of premalignant lesions [50]. These precancerous stages have been classified by the World Health Organization [51]. Severe dysplasias are usually grouped with in situ carcinomas in a group called “high-level

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precancerous” lesions, reflecting their significant tendency to become invasive. Classic treatments used for severe dysplasias and in situ carcinomas are stripping, external radiation, and laser resection. When work with the CO2 laser first began, some surgeons would use it to vaporize the mucous membrane, while others would perform a resection with histological margin examination. The first lasers had a spot that was around a milli­ meter in diameter, which did not allow for dissection of Reinke’s space. Resection inevitably took place in the superficial part of the vocal ligament, or even the musculature. Technological progress has enabled the spot to be reduced to 100 or 200 mm, allowing us to pass into Reinke’s space while retaining the vocal ligament. Supra- or subglottic extension requires correct visualization, with resection of the false cord in the event of extension towards the ventricle or a transversal section of the glottic musculature in the event of inferior extension. Contralateral or bilateral extension causes problems of anterior glottic synechia, altering the vocal quality. As with stripping, a resection carried out in stages can resolve this difficulty. Initially, we tend to treat the side which has been most affected, going up to the median line on the anterior commissure and 2 or 3 months later carry out resection of the contralateral side, slightly overlapping the median line. Table 25.5 presents the results of the three main treatment techniques: stripping, radiotherapy, and laser resection. The rate of recurrence after stripping is very variable, ranging from 10% to 60% according to different authors. It is higher than that seen with the two other techniques. After radiation, the local recurrence rate varies between 0% and 20%. Sadri compiled 605 patients who had undergone radiotherapy and reported a 12% recurrence rate [62]. Radiotherapy however, gives a specific survival rate of close to 100%, but a total laryngectomy rate of 10%. With the CO2 laser, the local recurrence rate is between 5% and 15%, averaging approximately 10%. The major advantage of the CO2 laser is that Table 25.5  Literature review concerning laser of “in situ” Number Technique Authors Year of patients Stripping Miller [52] 1971 100 Hintz [53] 1981 27 Stenersen [54] 1988 41 Radiotherapy

Laser

Pene [55] Elman [56] Le [57] Spayne [58] Garcia-Serra [59]

Steiner [8] Moreau [20] Eckel [25] Damm [60] Roedel [61] a Salvage –augmented local control rate

these recurrences can be treated in a nonmutilating way, either by repeating the laser resection or by radiation, with a rate of conservation of the larynx close to 100% and a specific survival rate also close to 100%. The surgeon-dependent nature of laser treatment is similar to that seen with invasive cancers. Small published series often show higher rates of recurrence, testimony to the importance of the surgeon’s skill [57, 63]. One of the major advantages of the laser is the ability to precisely classify the tumor. Patients receive radiotherapy on the basis of a biopsy which has shown a pre-cancerous stage while the lesion may in fact be microinvasive elsewhere. Laser resection enables classification of certain precancerous stages to be modified, based on analysis of the specimen, thus allowing for appropriate treatment following determination of the true extension of the tumor. An argument often advanced in favor of radiotherapy is that of vocal quality. The rate of salvage total laryngectomy after radiation renders this argument null and void. While laser resection is limited to the superficial part of Reinke’s space, the mucosal wave in stroboscopy is salvaged, with no consequences for the voice. However, minor vocal consequences often occur for various reasons: Reinke’s space cannot be detached, for example in case of hypertrophic laryngitis, deep biopsy resulting scarring, or in the event of subglottic, supraglottic, or contralateral extension.

Debulking of Airway Obstruction Pharyngeal or laryngeal cancers causing dyspnea require immediate restoration of a sufficient respiratory channel. The classic solution is a tracheotomy, carried out where required under local anaesthesia, prior to a total laryngectomy. The risk of recurrence around the orifice of the tracheotomy

Local recurrence %

Total laryngectomy %

5-year specific survival %

5-year overall survival %

25 63 46

– 19 –

–

– – –

– –

1976 1979 2000 2001 2002

79 69 54 67 30

15 17 18 1 10

10 14 13 1 10

1993 2000 2000 2000 2009

29 26 31 29 34

9 4 6 14 12

0 0 0 0 0

93a –

98 100 100

– – – 84 80

100 100 100 100 100

– 83 86 – 82

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is estimated at between 3% and 40%, particularly where the tracheotomy cuts across the tumoral tissue, but also as a result of neoplastic seeding [64]. Peristomial recurrences constitute an extremely unfavorable factor with a mortality rate of 80% to 90% [64, 65]. To avoid the need for tracheotomy, radical treatment can be proposed in the form of an emergency total laryngectomy [66]. The absence of an earlier assessment and practical contingencies, however, do not always permit this. A third possibility is the restoration of a sufficient airway passage by tumoral debulking using laser endoscopy [67]. The concept is simple; performing it is less so. Tumoral transsection often results in hemorrhage, which is difficult to control. The remaining tumor has a crumbly texture which can obstruct the airways again immediately. Subglottic extension, which is often dyspneal, makes this difficult to carry out and is further hindered by the presence of the ventilation tube. The ventilation jet is no simpler as an airway return has to be restored to avoid pulmonary complications. In the event of localization at the level of the laryngeal aditus, laser resection can result in aspiration. It is not rare to have to repeat endoscopic disobstruction. An endoscopic resection on the side which is not affected by the tumor occasionally helps. Debulking, however, most often enables a sufficient airway channel to be restored for a few weeks, allowing for the usual extended assessment to be carried out and for the curative procedure to be scheduled under the best conditions [6, 67]. Other types of laser techniques and different methodologies have been used with success, including the microdebrider [68].

Salvage After Glottic Radiation Failure Postradiation salvage surgery is difficult. The majority of T1–T2 glottic cancers are salvaged by total laryngectomy, with a failure rate of 20–50% [69]. In a minority of cases, open partial surgery is used, with a local recurrence rate of between 5% and 25% [70, 71]. Series published on salvage with laser resection are presented in Table  25.6. They are limited to a few dozen patients, demonstrating the small proportion of cases in which this laser surgery is possible. New recurrences can be seen in 25–60% of cases. Laser resection Table  25.6  Literature review concerning laser of carcinomas after radiotherapy Number Local Authors Year of patients recurrence % Quer [72] 2000 24 25 de Gier [70] 2001 40 58 Steiner [73] 2004 34 59 Ansarin [71] 2007 37 35

recurrent glottic Total laryngectomy % 25 50 21 30

can be repeated in the event of recurrence. The rate of total laryngectomy is between 20% and 50%. Some of these patients die from their cancer in the event of recurrence, but it is difficult to determine the exact proportion. All authors underline the technical difficulties of this laser resection in radiated areas as a result of imprecise margins.

Discussion Steiner uses the CO2 laser to carry out transoral resection of oral cavity cancers and those of the upper oropharynx, soft palate, or tonsillar area [6]. Other authors remain attached to conventional transoral instruments, such as the electric bistoury and “cold” instruments. They avoid the inconveniences of laser: coagulation necrosis at the margins, which renders scarring more difficult and increases the risk of stitches failing, and axial radiation complicating resection of the deepest part of the specimen. Histologic analysis of margins is more difficult with CO2 laser resection than with traditional means. A tissue thickness of 0.5 mm is destroyed by the laser section, reduced at best to 200  mm with Acuspot. The margins are subject to coagulation necrosis, which further complicates the analysis. When the laser was initially used, some authors advised the destruction of the tumor by vaporization without histological control of the margins. No significant series supports this concept. Formal or possible involvement of the resection margins of the specimen often occurs, in about 25–30% of cases [44, 45]. Re-resection for an inadequate margin uncovers residual tumor in only 20% of cases [45]. True histological involvement of the margins is associated with a higher rate of locoregional recurrence, an increased rate of distal metastases and a reduced specific survival rate [44, 45, 74]. Here we begin to appreciate all the difficulties of interpreting the histological involvement of the margins and the need to meticulously analyse them. Could a focal spread upon a margin be considered as insignificant following tissue destruction? Is an infra millimetric deep limit sufficient? Does the piecemeal resectioning technique enable as relevant an analysis of the margins as en bloc resection? Lymph node involvement in supraglottic and pharyngeal tumors often occurs. Bilateral neck dissection is indicated in the event of median or near-median tumor, and unilateral dissection in the event of a lateralized tumor. In the event of a microinvasive tumor, with less than 2  mm infiltration, the indication is more subtle. For the supraglottis, where lymph node involvement is rarer, a microinvasive tumor prompts simple monitoring. For the pharynx, the discussion remains open. If the tumor is clearly invasive, neck surgery can be carried out during the same operation, but can also be deferred. In the event of laser resection adjoining the cervical

25  Laser Endoscopic Treatment

region, it is preferable to delay neck surgery in order to avoid communication between the two operating areas and the risk of fistula formation. Mastering resection with the help of the CO2 laser is not easy. Most of the large series published have been carried out by a single surgeon, who has progressively enlarged the indications and who has acquired an optimal technique. Large published series give better results than the smaller ones. Teaching appears more difficult than with open surgery. This surgeon-dependent nature of the endoscopic approach limits its growth and results in a disparity of results. The term “laser” evokes new technology, which gives rise to great enthusiasm. In oncology, prudence imposes itself. In the past, various technologies such as color lasers and photosensitisers were tested with lukewarm results [75, 76]. The CO2 laser is only one methodology used in surgical section. Recent literature proposes the use of diode lasers, the KTP laser, and pulsed dye lasers [77, 78]. Coupling the CO2 laser with a robot appears to be a promising concept, which would facilitate the action of resection at the level of the base of the tongue and the supraglottis [79].

Conclusions Over the course of 30 years, driven by Wolfgang Steiner, the CO2 laser has become one of the means of treating early cancers of the upper aerodigestive tract. For dysplasias and in situ carcinomas, its results are better than those of radiation treatment, by decreasing the need for salvage total laryngectomy. For early glottic T1 and T2 cancers, removable endoscopically, it gives a specific survival rate of close to 100% with a total laryngectomy rate of around 2–3%, a rate less than that seen following radiotherapy. In the event of decreased mobility and fixation, the results are less favorable. Many authors prefer to perform open partial laryngectomy in these cases. For supraglottic cancers, the use of CO2 lasers requires significant expertise. No consensus exists on the indications. A local recurrence rate of approximately 10% is seen in T1 and T2 tumors and 20% for T3 and T4 tumors. These can most often be salvaged. The specific survival rate at 5  years is 80%, depending mainly upon the extent of distal and lymph node metastases. For pharyngeal cancers, indications also vary depending upon the authors. The role of metastasis and of second primaries is of clear dominance in determining outcome. The rate of local recurrence is 20% with a specific survival rate of 70% and an overall survival rate of 50%. Beyond simple morbidity, the fact that a tracheotomy can be avoided and that the postoperative course is simpler, the major advantage of laser resection remains the absence of locoregional dissemination. This allows for effective salvage

347 Table 25.7  Advantages and limitations of laser Advantages Limitations Global

More precise classification of T Avoid local dissemination Excellent salvage in case of local recurrence Postoperative course very simple Low cost

Required expertise Surgeon-dependent nature Inadequate endoscopic exposition

Glottic

For T1–T2a Local recurrence 10% Salvage total laryngectomy 2–3%

Involvement of anterior commissure Glottic fixation Low vocal quality in case of radical or bilateral cordectomy

Supraglottic

Avoid tracheotomy Local recurrence 15%

Disparity of contraindications T4, a part of T3 Extension near hyoid bone and to glottic area

Pharyngeal

Local recurrence 20% Very useful in case of multiple tumors

Controversy of indications and techniques Overall survival ~50%

Precancerous Local recurrence 10% Specific survival ~100% Avoid salvage total laryngectomy

2 stages in case of bilateral glottic extension

treatment to be carried out, whether by repeated laser resection, open surgery, or radiation. One inconvenience is the difficulty of learning this technique and hence its surgeondependent nature. Table 25.7 summarizes the advantages and the limitations of CO2 laser use.

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Chapter 26

Multidisciplinary Management of Skull Base and Superstructure Tumors Giulio Cantù, Carlo L. Solero, Stefano Riccio, Sarah Colombo, and Madia Pompilio

Abstract  Malignant tumors of the paranasal sinuses are rare, accounting for only 3% of all the head and neck malignancies. As a consequence, no report of a randomized clinical trial about different treatments has been published, and the chance to perform such a trial is remote. However, the combination of surgery and (chemo)-radiotherapy seems to offer better local control than radiotherapy alone. The treatment of skull base tumors is, by definition, a multidisciplinary work. Even in cases were surgery may be the only treatment, at least a neurosurgeon and a head and neck surgeon must collaborate to reach good results avoiding complications. Plastic and reconstructive surgeons, radiologists, anesthesiologists, critical care and rehabilitations experts, and nurses are also indispensable. Moreover, the quite steady indication for pre- or postoperative (chemo)radiotherapy involves the involvement of medical oncologists and radiotherapists in the therapeutic team. This chapter will demonstrate in details the above-­ mentioned principles, mentioning the more recent papers on this topic and my own large experience in the treatment of malignant skull base tumors. Moreover, I will take into ­consideration the most frequent histologic types and their different etiology and standard or experimental treatment. Keywords  Sinonasal cancer • Skull base • Paranasal sinus • Reconstructive surgery

Introduction Malignant tumors of the paranasal sinuses account for only 2–3% of the head and neck carcinomas and about 0.5% of all malignancies [1]. The low incidence and great variety of histologic types means that there are no large studies on G. Cantù (*) Department of Cranio-Maxillo-Facial Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, National Cancer Institute, Via Venezian 1, Milano 20133, Italy e-mail: [email protected]

management of these tumors. No randomized clinical trials about different treatments have been published, and the chance to perform such a trial is remote.

Etiology A possible occupational etiology of sinonasal cancers was first hypothesized in 1890, when a maxillary tumor was detected in a worker exposed to chrome [2]. An increased risk of sinonasal cancer has been demonstrated among workers exposed to formaldehyde, nickel, and chrome. Tobacco and alcohol are not considered major risk factors, even though heavy smokers have an increased risk of squamous cell carcinoma (SCC) [3]. The most interesting paranasal sinus tumor, for which there is an indisputable occupational etiology, is ethmoid adenocarcinoma, mainly Intestinal Type Adenocarcinoma (ITAC). Wood dust exposure as a risk factor for sinonasal cancers was recognized in 1968 by Acheson et al. [4]. Several papers have since been published on the occupational etiology of these tumors [5–9], including leather dust as a major risk factor [8]. These papers have a possible bias for properly classifying the histology and site of tumor. Some of the studies use the generic terms “nasal cancer” [4, 8], “nasal and sinonasal cancer” [9], and “sinonasal cancer” [3, 7]. Only one paper correctly stated in the title both the histology (adenocarcinoma) and location of the tumor (ethmoid sinus) [6]. Hadfield [5], who analyzed 92 patients with sinonasal cancer (34 SCC, 35 adenocarcinomas, and 23 anaplastic carcinomas), found that the tumor appeared to originate in the ethmoid sinus in all 35 patients with adenocarcinoma. This fact was confirmed in a later paper [10]. We carefully assessed the histology and tumor origin of 499 patients with sinonasal malignant tumors treated at the Istituto Nazionale dei Tumori of Milan between 1987 and 2001 [11]. Of the 249 patients with ethmoidal tumors, 124 (49.8%) had adenocarcinoma, and 90.4% of the adenocarcinoma patients had a history of wood or leather dust exposure. Of the remaining 125 patients with ethmoidal tumors

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_26, © Springer Science+Business Media, LLC 2011

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other than adenocarcinoma, only 2 (1.6%) had been exposed to these dusts. The remaining 250 patients had non-ethmoidal sinonasal tumors, 17 of which (6.8%) were adenocarcinomas. No wood or leather dust exposure was reported in any of these patients. The different role of hardwoods and softwoods in tumor development remains largely unknown. Some authors [9, 12] in Northern Europe (where furniture industries employ mainly softwoods) highlighted a minor and different carcinogenetic power of softwoods compared to the hardwoods that are more often employed in Southern Europe. The authors found an association between hardwood dust exposure and adenocarcinoma, while softwood dust exposure alone was associated with epidermoid and anaplastic carcinomas. However, there is a general consensus about the danger posed by a working environment with over 5 mg/m3 of wood dust, while some [13] suggest a lower dust level of 1 mg/m3. Sinonasal tumors treated with an anterior craniofacial resection had different histological compositions in Europe compared to North America. The rate of adenocarcinomas in European countries is very high: Roux [14] (France) 74%, Suarez [15] (Spain) 53%, Cantu [16] (Italy) 49%, Cheesman [17] (United Kingdom) 27%. Conversely, the rates are much lower in American studies: McCutcheon [18] (USA) 17%, Bentz [19] (USA) 12%, Donald [20] (USA) 6%, and Irish [21] (Canada) 5%. Bridger [22] (Australia) reports 37% of sinonasal tumors were adenocarcinoma, consistent with the UK study. While no definitive explanation for these discrepancies exists, Blot et al. [23] hypothesizes the following: • While the commonly accepted threshold for the level of wood dust in the air is 5 mg/m3, many European artisan furniture factories and joinery may have exceeded that threshold. • Hardwoods, which are more dangerous than softwoods, are probably more widespread in Europe than in America. • America adopted safety measures, such as masks and aspiration devices, earlier than Europe. Given that these tumors have a latent period of about 30–40 years between the beginning of exposure and clinical presentation [24], there will probably be a reduction in the incidence of this disease in Europe over the next decades resulting from the improved factory conditions. Polymorphisms in xenobiotic metabolizing enzymes play an important role in the gene–environment interaction and may contribute to a high degree of variance in the individual susceptibility to cancer development. The Experimental Molecular Pathology Unit of our Institution investigated the role of polymorphisms in the CYP1A1 and GSTM1 genes in 30 ethmoid ITAC patients and 79 healthy blood donors [25]. The results revealed that patients with the CYP1A1 codon 461 polymorphism may be at an increased risk of developing ITAC, and that this risk increases in the presence of both the

G. Cantù et al.

polymorphism at this CYP1A1 codon and the GSTM1-null genotype. Our study strongly suggests that these genotype investigations may be useful in determining the exposed individuals who are at risk for developing ethmoid ITAC.

Pathology and Natural History Excluding the nasal cavity, the maxillary sinus is the most frequent site of tumor origin (70–75%), followed by the ethmoid sinus (20–25%). Primary tumors in the frontal or sphenoid sinus are unusual, even though these sinuses are sometimes involved in large neoplasms. The most common histologic type in maxillary sinus is SCC, more or less differentiated, followed by tumors affecting minor salivary glands: adenocarcinoma (ADE), adenoid cystic carcinoma (ACC), and mucoepidermoid carcinoma (MEC). Sarcomas (malignant fibrous histiocytoma, malignant peripheral nerve sheath tumors, chondro-, osteo-, fibro-, leiomyo-, and angiosarcomas) are rare. Rhabdomyosarcomas are more frequent, particularly during childhood [26]. In the ethmoid sinus, in addition to SCC and ITAC, other common histologies include sinonasal undifferentiated carcinomas (SNUC), melanomas, and sinonasal neuroendocrine tumors: esthesioneuroblastomas (ENB), neuroendocrine carcinomas (NEC), and small cell carcinoma neuroendocrine type (SCCNET) [26]. These tumors often grow silently, meaning that patients often present with advanced-stage disease. The air-filled sinus cavities do not offer resistance to tumor growth, and the tumor becomes symptomatic only after it erodes the bony walls. A tumor of the maxillary sinus may affect the hard palate and alveolar ridge inferiorly, the orbit superiorly, the cheek anteriorly, or the pterygoid plates posteriorly. These tumors may also invade the pterygopalatine and infratemporal fossa, the greater wing of the sphenoid and the middle cranial fossa. A tumor of the ethmoid may spread inferiorly into the nasal cavity, laterally into the orbit, posteriorly into the sphenoid sinus and nasopharynx, or superiorly into the anterior cranial fossa (after eroding the cribriform plate). The medial periorbita provides an effective barrier against tumor invasion, but the natural holes in this structure (lacrimal duct, anterior and posterior ethmoidal arteries) are roads for tumor invasion into the orbital contents. Although the dura is resistant to tumor growth, the olfactory nerves allow for the tumor to spread intradurally. The lymphatics from the anterior part of maxillary sinus drain through the facial lymphatic vessel into the nodes at level I and II. Lymphatics from the ethmoid and posterior part of maxillary sinus drain into the lateral retropharyngeal nodes, which lead to the deep cervical chain. Neck metastases are an unfavorable prognostic factor [27–33], although the incidence of neck metastases at

26  Multidisciplinary Management of Skull Base and Superstructure Tumors

presentation is low. The meta-analysis by Dulguerov et al. [27] reported about 12% of patients presented with neck metastases, although the incidence of nodal metastases during the follow-up period was around 13%. In a previous paper [34], we reviewed the medical records of 704 consecutive patients surgically treated for paranasal sinuses malignant tumors (305 ethmoid sinus tumors and 399 maxillary sinus tumors). Nodal metastases from ethmoid tumors were very rare, both at presentation (1.6%) and during follow-up (4.3%). Moreover, the majority of subsequent neck metastases appeared with a recurrence of the primary tumor. Only patients with SNUC, NEC, or SCCNET had a high rate of regional recurrence (25%), and these tumors probably behave similarly to nasopharyngeal undifferentiated carcinoma in this regard. In the maxillary sinus, the rate of neck metastases for non-squamous cell carcinomas was very low at presentation (6%). Subsequent nodal metastases were rare, except in SNUC (13%) and ADE (22.2%). Actually, ADE of maxillary sinus originates in the minor salivary glands and acts like ADE of the major salivary glands. This type of cancer is very different from ITAC of the ethmoid sinus, which rarely metastasized regionally. Only 16 of the 156 patients with SCC of the maxillary sinuses presented with nodal lesions (10.3%), of which eleven patients were staged as T2, one was as T3, three were considered T4a and one was stage T4b. All five of the patients with T3–T4 tumors had involvement of the oral mucosa as well. Four of the 26 (15.4%) patients with SNUC presented with nodal lesions, as did 13 of the 217 (6%) patients with other histologic types. These data strongly suggest that a higher percentage of stage T2 tumors present with cervical metastases than stage T3–T4 tumors. By definition, a T2 tumor involves the floor of maxillary sinus (with possible mucosal invasion of the hard palate and upper gum) and/or the inferior nasal cavity, both of which have a more expansive lymphatic network than the mucosa of paranasal sinuses. Therefore, in terms of lymph node metastases, these tumors have a behavior more similar to oral cancers than paranasal cancers. The fact that paranasal sinus carcinoma behaves differently than other head and neck carcinomas was first recognized in 1937 by del Regato [35] and has been confirmed by numerous authors [27, 32].

Symptoms While tumors affecting the hard palate or nasal cavity may cause symptoms early, superstructure tumors are asymptomatic for a long time, making an early diagnosis difficult. When the tumor is located in the ethmoid sinus or the upper part of the nasal cavity, patients may present with only unilateral nasal obstruction. Epistaxis generally

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occurs only in vascular tumors (hemangiopericytoma and esthesioneuroblastoma), with the patient often complaining of blood-stained secretions. More advanced tumors that have invaded the nasolacrimal duct and orbit may cause epiphora, proptosis, and diplopia. The tumor may also invade the orbital apex posteriorly, causing ophthalmoplegia and visual loss, the sphenoid sinus and the nasopharynx. Anterior involvement of the nasal bones produces a characteristic broadening of the upper nasal region. Although few patients report anosmia as a first symptom, almost all patients remember some loss of smell when specifically asked. Incredibly, tumor invasion of the anterior cranial fossa is generally silent. Invasion of the infraorbital nerve, leading to dysesthesia and pain at the level of the cheek and upper lip, is often misdiagnosed as trigeminal neuralgia. A tumor invading the infratemporal fossa may infiltrate the third branch of the fifth cranial nerve at the foramen ovale, causing disesthesia, pain and anesthesia of the chin, inferior teeth (mandibular nerve), and omolateral tongue (lingual nerve). In our series, some patients had these symptoms for over a year prior to diagnosis, particularly with slow-growing tumors like adenoid cystic carcinoma. A tumor can infiltrate the pterygoid muscles, causing trismus, and it may also erode the greater wing of the sphenoid, spreading into the middle cranial fossa.

Staging Establishing a consistent prognostic staging system for each extension of paranasal sinus carcinoma has proven difficult, as demonstrated by the numerous classification schemes previously published [36–41]. All systems only considered tumors of the maxillary sinus and assigned a higher stage for tumors with posterosuperior extension. In 1906, Sebileau [42] realized that the prognosis of tumors differed depending on their location, either inferiorly or superiorly in the paranasal sinuses. He divided the upper jaw with two horizontal parallel “imaginary lines” into “infrastructure, mesostructure and suprastructure”. In 1933, Öhngren [43] recognized that Sebileau’s system did not address posterior extension of the tumor and proposed a classification system based upon a hypothetical plane passing through the inner canthus and the mandibular angle. The “malignancy plane” divided the upper jaw into an infrastructure (“topographically more benign tumors”) and a suprastructure (“tumors of more malignant character”). Öhngren’s line was the basis for the division between T1–T2 and T3–T4 maxillary sinus carcinomas in the first four versions of the American Joint Committee on Cancer (AJCC) classifications [44–47]. According to these guidelines, the maxillary sinus was “the only site to which the following classification applies. The ethmoid sinus and nasal cavity may ultimately be defined similarly with further study.”

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While the AJCC partially staged paranasal sinus carcinoma from the beginning, the International Union Against Cancer (UICC) did not stage paranasal sinus tumors in the first three editions of its manuals. A classification of maxillary sinus carcinoma, similar to that of the AJCC, appeared only in the fourth edition in 1987 [48]. Several studies of the AJCC-UICC classification of maxillary sinus carcinoma have demonstrated its prognostic value, with a progressive worsening of the prognosis from T1 to T4. The absence of a universally accepted classification of ethmoid cancer led to an obvious lack of disease staging in the literature. Sisson [49] wrote: “The ethmoid cancers were not staged because there is no generally accepted staging system for this site.” Similarly, after having staged tumors of the maxillary sinus, Spiro [50] wrote: “As there is no widely accepted staging system for the remaining sinuses or the nasal cavity, no attempt was made to stage tumors arising in these sites.” In fact, some authors have tried to stage nasoethmoid tumors. Kadish [51], Biller [52], and Dulguerov [53] proposed a classification for esthesioneuroblastomas. Ellingwood [54] and Roux [55] published a classification for tumors of the nasal cavity and ethmoid–sphenoid sinuses. Despite of their historical significance, these classification systems were never tested in large-scale studies to determine their prognostic value. In 1997, the fifth edition of both the AJCC and UICC guidelines contained an unambiguous staging system for cancers of the maxillary sinus, nasal cavity and ethmoid sinus. Even if the terms “infrastructure” and “suprastructure” formally disappeared, the concept of tumors divided by Öhngren’s line having differing prognosis was present in the fifth and sixth edition of the AJCC-UICC guidelines. In the absence of a universally accepted staging system, we presented in 1997 an original classification for malignant ethmoid tumors [56] based on the most commonly accepted prognostic factors, including involvement of dura mater, intradural extension, involvement of the orbit (particularly the apex), invasion of maxillary, frontal and/or sphenoidal sinus, and invasion of the infratemporal fossa and skin (Table 26.1). In 2005, we tested our original classification for ethmoid tumors (INT – Istituto Nazionale Tumori) in terms of prognostic performance versus the fifth and sixth AJCCUICC classifications [57–60] (Table  26.2). Both the 1997 and 2002 AJCC-UICC classification systems seemed to have limited prognostic value. In contrast, our INT classification demonstrated the progressive worsening of prognosis with different tumor classes for the overall series, when applied separately to untreated and recurring cases, and when applied only to adenocarcinomas, the most frequent histologic type in our series [61]. We agree with Dulguerov et al. [62], who stated in a recent review that “while the evolution of TNM staging is a work in continuous progress, the T staging of ethmoid and nasal primaries needs an urgent revision.”

G. Cantù et al. Table 26.1  INT classification of ethmoid tumors T1 Tumor involving the ethmoid and nasal cavity but sparing the most superior ethmoidal cells T2 Tumor with extension to, or erosion of, the cribriform plate, with or without erosion of the lamina papyracea and without extension into the orbit T3 Tumor extending into the anterior cranial fossa extradurally and/or into the anterior two-thirds of the orbit, with or without erosion of the anteroinferior wall of the sphenoid sinus, and/or involvement of the maxillary and frontal sinus T4 Tumor with intradural extension, or involving the orbital apex, the sphenoid sinus, the pterygoid plate, the infratemporal fossa or the skin

Table  26.2  AJCC-UICC-2010 classification of nasal cavity and ethmoid sinus T1 Tumor restricted to any one subsite, with or without bony invasion T2 Tumor invading two subsites in a single region or extending to involve an adjacent region within the nasoethmoidal complex, with or without bony invasion T3 Tumor extends to invade the medial wall or floor of the orbit, maxillary sinus, palate, or cribriform plate T4a Moderately advanced local disease. Tumor invades any of the following: anterior orbital contents, skin of nose or cheek, minimal extension to anterior cranial fossa, pterygoid plates, sphenoid or frontal sinuses T4b Very advanced local disease. Tumor invades any of the following: orbital apex, dura, brain, middle cranial fossa, cranial nerves other than (V2), nasopharynx, or clivus Used with the permission of the American Joint Committee on Cancer (AJCC), Chicago, IL. The original source for this material is the AJCC Cancer Staging Manual, Seventh Edition (2010) published by Springer Science and Business Media LLC, www.springer.com

Imaging All patients with a malignant tumor of the superstructure must undergo a high-resolution contrast-enhanced computer tomography (CT) in axial and coronal planes, and/or a multiplanar magnetic resonance imaging (MRI) enhanced with gadolinium. CT is helpful in determining the erosion of the bones surrounding the paranasal cavities and the involvement of the skull base. Although CT soft tissue windows are essential to evaluate intracranial or intraorbital extension of the tumor, MRI allows a better distinction of tumor from the adjacent soft tissue (orbital contents, dura, brain, cavernous sinus, infratemporal fossa, and carotid artery). Although neck nodes metastases are unlikely for these tumors, CT or MRI must be extended to the neck, and chest CT or positron emission tomography (PET scan) may be useful to exclude distant metastases.

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Histologic Diagnosis A biopsy is mandatory for a histologic diagnosis. The biopsy must be made in representative tissue, avoiding necrotic vegetations. An endoscopic approach, sometimes in the operating room, is almost always sufficient for a proper biopsy. An open procedure should be avoided, except in cases where an endoscopic approach is impossible. We recommend all histologic slides be read separately by two experienced pathologists. Cohen et al. [63] report a high rate of misdiagnosis of olfactory neuroblastoma, as the diagnosis was changed by the pathologists at the M.D. Anderson Cancer Center in 10 out of 12 cases. In our experience, the rate of changing diagnosis is not as high, with our pathologist changing the previous diagnosis in about 20% of cases. Because the treatment regimens and prognosis of tumors types are often significantly different, the correct diagnosis should be confirmed before initiating treatment.

Prognostic Factors Significant prognostic factors include the histologic findings of the primary tumor, the presence of neck nodes metastases, the status of surgical margins, and the extent of intracranial and intraorbital involvement. Tumor histology is statistically related to outcome. Patients with mucosal melanoma and undifferentiated carcinoma (SNUC, NEC, and SCCNET) have the worse outcome, whereas those with minor salivary gland tumors, esthesioneuroblastomas, and low-grade sarcomas have the best outcomes [27, 64]. However, one must remember that tumors like adenoid cystic carcinoma and esthesioneuroblastoma may recur after a long period of time, so the common reported 5-year survival may be misleading. Patients with ITAC have a better disease-specific survival than those with squamous cell carcinoma [34, 64, 65]. Although neck metastases are rare, their presence, whether upon presentation or later, worsens the prognosis for patients with ethmoid and maxillary sinus tumors. In the ethmoid sinus group of our series, 5-year survival rates were 45.3% in patients with N0 tumors versus 0% in those with N+ (N1, N2, or N3) tumors. In the maxillary sinus group, the corresponding 5-year survival rates were 50.6% and 16.8% [34]. No patients with ethmoid malignant tumors and nodal metastases, either at presentation or during follow-up, survived. For patients with maxillary sinus tumors, the situation was similar, although slightly less dramatic. Because local failure is the most common cause of death, the status of surgical margins is an important prognostic factor [64, 66, 67]. As tumors of the superstructure often involve

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the skull base and orbit, patients with these extensions have a worse prognosis. Therefore, craniofacial surgical techniques are mandatory to try to reach negative surgical margins.

Treatment The treatment of tumors of the superstructure is by definition a multidisciplinary field. A wide variety of management approaches have been advocated and practiced in the past, and there is currently no standard treatment. The most common approaches involve some combination of surgery, radiation, and chemotherapy. The timing and combination of these three therapeutic means is dictated by the histology, locations, and extensions of the tumor. As surgery often entails a craniofacial resection, this treatment may also be dictated by the expertise of the surgical team. Given the rarity of malignant tumors of paranasal sinuses, particularly tumors of the superstructure, the retrospective studies published by individual institutions are often based on a small number of patients with a diversity of histologic findings and tumor extension. Most studies also have selection bias, as higher proportions of patients with favorable lesions are found mainly in the surgery groups, whereas most patients with advanced disease, unfavorable histology, and/ or unresectable tumors are found in the (chemo) radiation groups. Nevertheless, the combination of surgery and (chemo)radiotherapy seems to offer better local control than radiotherapy alone, as “the meta-analysis confirmed that surgery and combined surgery and radiation offer better local control and cure rates than radiotherapy alone” [27]. Another study concluded that “surgery and postoperative radiation therapy may result in improved local control, absolute survival, and complications when compared with radiation therapy alone” [28]. Surgery and postoperative (chemo)-radiotherapy is considered the treatment of choice in most centers, even if some continue to prefer primary radiotherapy [68].

Surgery For many years, surgical treatment of paranasal sinus cancers remained little more than a piecemeal resection. Lizars of Edinburgh, in 1826, proposed entirely removing the superior maxillary bone, and performed the first resection in 1829. He accurately described the procedure, although he could not remove the posterior portion of the tumor around the pterygoid process [69]. The goal of surgery is complete, en bloc resection of the malignant tumor with negative margins. Unfortunately, given the frequent extensions of these

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tumors into the orbit, infratemporal fossa, middle and/or anterior cranial fossa, tumors involving the superstructure were considered unsuitable for a radical resection until the 1960s. Innovative surgical approaches into the pterygomaxillary and infratemporal fossa for tumors with posterior extension were introduced by pioneers such as Conley [70] and Crockett [71]. In 1970, Dingman and Conley [72] wrote, “In the standard maxillectomy, the posterior chisel cut is made in the pterygomaxillary sulcus, thus freeing the ­maxilla from the lateral process of the pterygoid lamina. Examination of a skull shows that this margin is inconsistent with good tumor management for many maxillary cancers with posterior extensions. Failure at this margin is often responsible for failure to effect local control of the maxillary cancers, and has led many clinicians away from surgery as a method of primary treatment. The obvious extension of the maxillectomy operation is the inclusion of the pterygoid plates and muscles to form the posterior margin of the specimen. When the surgeon attempts this by the anterior, or Weber-Fergusson approach, he finds that he must develop this critical margin in a cavity filled with blood, within several mm of the internal carotid artery”. After this publication, the anterolateral approach became the standard treatment for tumors involving the pterygomaxillary fossa. Similarly, paranasal sinus tumors invading the skull base (middle or anterior cranial fossa) continued to be considered unresectable. Some isolated reports in the 1950s discussed a craniofacial approach to tumors of the frontal sinus [73, 74]. However, in 1963, Ketcham [75] was the first to report a remarkable series of patients with tumors involving the anterior skull base who were treated with a combined transcranial and transfacial approach. Today, anterior craniofacial resection is the standard treatment for these tumors, and the prognostic factors have been quite well established [76, 77]. Ketcham, a head and neck surgeon, began his enterprise with Van Buren (a neurosurgeon) and they published articles [78] stressing the importance of this collaboration: “Although some may consider a neurosurgeon helpful but not necessary for this surgical undertaking, his preoperative evaluation and intraoperative handling of the skull, dura, and sometimes the brain contributes to a lower rate of complications and a greater cure rate.” Given the concept of a double approach (Fig. 26.1), surgical resection must be tailored to the tumor’s specific extension. For an ethmoid tumor involving the anterior cranial fossa but sparing the maxillary sinus and the orbit, a total ethmoidectomy with medial maxillectomy is the standard treatment. The anterior and inferior walls of the sphenoid sinus and the lamina papyracea must always be removed to allow for a radical resection (Fig.  26.2). For tumors with intracranial extension, the dura should always be resected and reconstructed, especially when infiltrated. Intradural invasion is usually a contraindication for surgery. In our series of anterior craniofacial resections for superstructure

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Fig. 26.1  Our standard coronal and lateral rhinotomy incisions for an anterior cranio-facial resection. The lateral rhinotomy incision without lip-splitting provides adequate exposure for total ethmoidectomy and medial maxillectomy

Fig. 26.2  Sagittal postoperative MR image showing total ethmoidectomy with resection of the anterior wall of the sphenoid sinus

malignant tumors, only some patients with esthesioneuroblastomas and intradural extension survived [61]. When the tumor has invaded the lamina papyracea, the medial periorbital layer should be resected, even if it is uninvolved. An orbital exenteration is required if the tumor has invaded the orbit deeply. Sometimes the orbital contents may be preserved, but the medial and inferior walls of the orbit must be removed. In these cases, alloplastic materials and free bone grafts are not a good choice for reconstruction as most of these patients will undergo postoperative radiotherapy, with probable extrusion of these materials. In spite of reconstruction with vascularized flaps, patients may complain of possible dysfunction of the eye, especially if postoperative radiotherapy is used [79].

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with intestinal type adenocarcinomas. As mentioned above, ITAC is a professional disease involving the whole ethmoid sinus. As the metaplastic transformation of ethmoidal mucosa to enteric-type epithelium precedes the development of enteric adenocarcinoma [83, 84], pre-neoplastic or neoplastic foci may be present in macroscopically uninvolved sites of ethmoid. In some cases, we found small tumor localizations in the contralateral ethmoid. These foci were separate from the apparent primary tumor and undetected by CT, MRI, and PET. As ITAC is a locally aggressive tumor that easily infiltrates the underlying bone [85], we believe that a total ethmoidectomy must always be performed. If this can be achieved endoscopically, we welcome such an approach. Fig.  26.3  Anterolateral craniofacial resection for a superstructure tumor involving both anterior and middle cranial fossa. After the resection of the tumor, the surgical instruments, introduced through the transfacial and anterior transcranial approaches, cross in the infratemporal fossa where the temporal lobe is exposed

For tumors involving the anterior cranial fossa, the posterior wall of the maxillary sinus, and the infratemporal fossa, a combined anterior craniofacial and infratemporal approach is mandatory, as the pterygoid plates and muscles must be removed. Sometimes the greater wing of the sphenoid may also be infiltrated requiring an anterolateral craniofacial resection [80] (Fig. 26.3). Following these resections, especially orbital exenterations, a vascularized free flap is required for a good functional and cosmetic reconstruction. In the last decade, a number of papers have discussed endoscopic resection of malignant tumors involving the anterior skull base. While most include a small number of patients with brief follow-up, one paper from two Italian university hospitals report a number of purely endoscopic resections for T1–T2 ethmoid adenocarcinoma (12 cases) and squamous cell carcinoma (4 cases), with a median followup of 47  months. The 5-year disease-specific survival of these patients was 93% [81]. In a following paper from the same institutions, 134 patients were treated with an endoscopic approach alone while 50 cases used a combined cranioendoscopic approach [82]. The 5-year disease-specific survival was 91% for patients treated with endoscopic approach and 58% for those who underwent cranioendoscopic resection. Tumor size differed between the two groups, with the larger tumors requiring the cranioendoscopic approach. Nevertheless, the authors conclude that when properly planned and completed by an experienced surgeon, endoscopic surgery is a valid alternative to standard surgical approaches for the management of selected malignancies of the sinonasal tract. We believe that regardless of method, resection of the sinonasal component of the tumor must be radical, especially

Radiotherapy There are few large-scale studies of patients with paranasal sinus malignant tumors treated with radiotherapy alone, and especially superstructure tumors. The majority of papers looked at a small number of patients, and included those who received postoperative radiotherapy and those treated with radiotherapy alone, but received surgery if radiotherapy failed. Almost all these papers reported combined surgery and radiotherapy worked better than RT alone. Katz et  al. [86], discussing their experience in treating malignant tumors of the nasal cavity, and paranasal sinuses (excluding the nasal vestibule and the maxillary sinus), state that “until approximately 17 years ago, most nasal cavity and paranasal sinus tumors were treated with high-dose irradiation alone at the University of Florida. On the basis of our experience, a change in treatment philosophy has occurred such that most patients undergo resection followed by postoperative irradiation. Surgery alone may be acceptable for very early cancers of the nasal cavity. Radiation therapy alone is used in patients with unresectable disease.” Another paper on sinonasal undifferentiated carcinoma from the same institution makes a similar statement: “Our current guidelines are to treat patients with apparently resectable tumor with craniofacial resection and postoperative RT” [87]. Guntinas-Lichius et al. [88] report on 229 patients with nasal and paranasal sinuses cancer treated at a single institution. Although the study suffers from selection bias, patients treated surgically had higher overall survival rates than patients who only received radiotherapy. The multivariate analysis for overall survival revealed that the type of therapy was an independent risk factor. Surgery combined with radio(chemo)therapy achieved better results in comparison to radio(chemo)therapy alone. Tanzler et  al. also wrote that “one advantage associated with combined surgery and RT is that it may be possible to reduce the RT dose and thus reduce the risk of RT-induced optic neuropathy” [87]. Regarding the use of modern

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radiotherapy techniques in the postoperative setting, the same authors wrote “Hyperfractionated RT is employed to further reduce the risk of injury to the visual apparatus. Intensity modulated radiotherapy (IMRT) and/or proton beam therapy may be useful to produce a more conformal dose distribution to reduce the dose to normal tissues and, thus, late toxicity” [87]. A paper from the Memorial SloanKettering Cancer Center draws a similar conclusion: “Complete surgical resection followed by adjuvant RT is an effective and safe approach in the treatment of paranasal sinus cancer. Emerging tools, such as three-dimensional conformal treatment and, in particular, intensity-modulated RT for paranasal sinus tumors, may minimize the occurrence of late complications associated with conventional RT techniques” [89]. Regarding neutron radiotherapy, Douglas et al. [90] report a 5-year actuarial local-regional control of 59% of tumors that do not involve the cavernous sinus, base of skull, or nasopharynx. The local-regional control was significantly lower for patients with tumors involving these sites (15%). In a following paper [91], the same authors stated that “variables associated with decreased local-regional control in the patients with gross residual disease as determined by multivariate analysis included base of skull involvement and biopsy only versus an attempted surgical resection prior to treatment.” For proton beam radiation, Pommier et al. [92] concluded that tumor involvement of the sphenoid sinus and clivus are adverse prognostic factors. In a review on proton therapy in clinical practice, Brada et al. [93] concluded that “the lack of available evidence in favor of protons does not mean that protons may not be useful in selected tumors. It should be a stimulus for more research, particularly in the form of appropriately designed and powered prospective studies.”

Chemotherapy Chemotherapy alone is normally reserved for cancers of the superstructure that are too advanced to be treated by surgery or radiotherapy, patients presenting with metastatic disease, or recurrent disease. However, chemotherapy may have a role into complex multimodal treatment plans along with surgery and radiotherapy. Beginning in 1970, some Japanese authors reported high cure rates with a combination of intra-arterial chemotherapy with 5-fluorouracile (5-FU), necrotomy, and radiotherapy. Using these treatment combinations, Sato et  al. [94] and Sakai et al. [95] achieved a 5-year cumulative survival rate of 67% and 54%, respectively. Other Japanese authors were unable to reproduce these results, concluding that the addition of intra-arterial chemotherapeutic agents to either surgery

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[96] or radiotherapy [97] did not improve survival. Shibuya et  al. [98] ascribed these contradictory results to the fact that the maxillary tumors receive blood from not only the internal maxillary artery, but also the facial and ethmoidal arteries. These latter arteries arise from the internal carotid artery and are the main feeding vessels of tumor of the ­ethmoid. This diverse blood flow may cause an irregular ­distribution of the intra-arterially infused antimetabolites, leading to decreased effectiveness. In order to prevent this situation, physicians have combined superselective intraarterial chemotherapy with radiotherapy and surgery in the last decade. Studies of this treatment method have reported a 5-year survival of 75% and 53% [99, 100]. Knegt et al. reported an interesting experience using surgical debulking, low dose of irradiation, topically applied cytostatic drug (5-FU), and necrotomy [101]. The actuarial 5-year survival rate for squamous cell carcinoma and undifferentiated carcinoma of the maxillary sinus was 52%, and 100% for patients with adenocarcinoma of the ethmoid sinus. A subsequent paper by the same authors reported their experience in treating 62 patients with ethmoid adenocarcinoma. They performed surgical debulking via an extended anterior maxillary antrostomy followed by a combination of repeated topical chemotherapy (fluorouracil) and necrotomy. Eight patients (13%) required additional radiotherapy for local recurrence, while one patient required surgery for regional lymph node metastases. Adjusted diseasefree survival at 10 years was 74% [102]. However, we only know of one other reported on this approach to ethmoid adenocarcinoma [103]. There are few reports on the use of systemic chemotherapy in paranasal sinus carcinoma, and sinus squamous cell cancer is not included in many head and neck prospective randomized trials on chemotherapy and/or radiotherapy. The most often applied schedules were platinum-based, with a response rate ranging from 36% to 84% [104]. The combination of primary chemotherapy, surgery, and postoperative (chemo)radiotherapy achieved very high cure rates [105], particularly in cases with pathologic complete remission (pCR) after neoadjuvant chemotherapy [106, 107]. The ability to predict complete response to primary chemotherapy by analyzing predictive biomarkers, such as TP53, is critical to determining the usefulness of chemotherapy. In our institution, 30 patients with ethmoidal ITAC have been enrolled in a phase II study using cisplatin, fluorouracil, and leucovorin (PFL) followed by craniofacial resection and radiation. On surgical specimens, absence of viable tumor cells was defined as pCR. The TP53 status and p53 function, analyzed on pretreatment biopsies, were retrospectively correlated with pathologic results and patient outcome. In patients with wild-type (wt) TP53 or functional p53 protein, pCRs were seen in 83% and 80% of patients, respectively. However, only 11% of patients with mutated TP53 achieved

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pCR, whereas no patients (0%) with an impaired p53 protein had pCR. At a median 55-month follow-up, all pCR patients were disease-free, while 44% of nonresponsive patients experienced relapse. These results indicate that differences in TP53 mutational status or protein functionality strongly influence pathologic response to primary chemotherapy and ultimately prognosis. PFL seems to be highly effective in patients with a functional p53 protein, even when encoded by a mutated TP53 gene. However, ITAC patients carrying a dysfunctional p53 protein will not respond to PFL [108]. The fact that only 40% of ITAC patients have a functional p53 protein diminishes our enthusiasm over these results.

Conclusions The actual prognosis for malignant tumors of the superstructure is still difficult to determine. The reported 5-year local control and survival rates are somewhat unreliable, as these studies include patients with different histologies, localizations, extensions, and treatment strategies. In spite of the best modern treatments available, the prognosis of these tumors continues to be disappointing. Many patients present with advanced-stage tumors and with intracranial and intradural extension. In our study of ethmoid tumors [65], the prognosis of adenocarcinomas and esthesioneuroblastomas was better than for the other histologic types. In particular, the prognosis was very unfavorable for melanomas. Epidermoid carcinomas also had a poor prognosis due to a large number of undifferentiated types. Patients with adenoid cystic carcinomas had a good overall survival, but only a short diseasefree survival, as these patients may survive for a long time after local recurrence or lung metastases. Untreated patients had better results compared to patients with relapses after previous treatment, suggesting that for these tumors, the first treatment is often the only treatment. The cure rates of patients with a tumor involving the middle cranial fossa are very low. For these patients, we may only perform surgery to improve the quality of their remaining life [80]. In conclusion, we must employ multidisciplinary treatments for these tumors, and translational research must continue to help improve how and when such treatments are used.

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Chapter 27

Multidisciplinary Management of Oral Cavity and Maxillary Sinus Cancers Alexander D. Rapidis

Abstract  During the last 30 years the belief that oral/ head and neck cancer management is based on team work has been established. The functions of tumor boards and combined clinics is a common contemporary practice with an exceedingly large number of medical, surgical, and other specialties being part of comprehensive, multidisciplinary therapeutic head and neck teams. The basic treatment modalities remain surgery, radiotherapy, and chemotherapy. Basic surgical techniques have not changed dramatically over the last 30 years. Among the major changes are the variations in the surgical management of the neck of both clinically negative and clinically positive neck patients, as well as the management of the mandible especially in the early invasion of oral squamous cell carcinoma in the mandibular bone. The revolution in the surgical treatment of oral/ head and neck cancer is the introduction of reconstructive techniques with both pedicled locoregional flaps and free tissue transfer. These reconstructive techniques allowed for safer and wider resections with adequate disease-free margins and functional reconstruction of the created surgical defects. Contemporary radiotherapeutic treatment has very little similarities with that of the late 1970s. Modern technology with the institution of new forms of radiation and the application of sophisticated computerized methods have enhanced the therapeutic effectiveness of irradiation with an equal important reduction in the sparing in irradiation of normal surrounding tissues. This has led to an increased therapeutic dose in the tumorous site and a decreased severity of radiation-induced injuries. Alterations in the fractionations have also shown to produce better therapeutic results in selected cases. The era of methotrexate, the leading chemotherapeutic agent of the 1970s, was followed by the institution of platinumbased chemotherapies with or without the addition of 5 Fu.

A.D. Rapidis (*) Eastman Dental Institute, University College London, London, UK and Department of Head and Neck/Maxillofacial Surgery, Greek Anticancer Institute, Sant Savvas Hospital, Athens, Greece e-mail: [email protected]

Adjuvant and neoadjuvant schemes coupled with pre- or postoperative radiotherapy started in the late 1980s and showed a distinct survival benefit over radiotherapy alone. This major breakthrough was followed by the institution of various and diverse chemoradiation regimes tested over a large time period for their survival benefits. The introduction of taxanes and the development of molecular targeted therapies during the last 5 years have revolutionized the concept of chemoradiation. Induction chemotherapy and chemoradiation coupled with epidermal growth factor receptor antagonists proved to have a survival benefit in patients with locally advanced or recurrent squamous cell carcinoma of the head and neck. Other biological agents against tumor angiogenesis or restoring cell apoptosis are being tested in various phase I or II trials. Perhaps the most promising noninvasive therapeutic method for squamous cell carcinoma of the oral mucosa is immunotherapy. The clinical applications so far are very limited but the research into these pathways is vast and extended. Keywords  Oral squamous cell carcinoma • Head and neck tumors • Oral cavity cancer • Head and neck cancer • Treatment of the oral cavity cancer • Maxillary carcinoma • Chemoradiation • Induction chemotherapy • Targeted therapies • Combined treatments

Introduction Cancer of the oral cavity comprises nearly 30% of all malignant tumors of the head and neck. Squamous cell carcinoma represents approximately 90% of the cases [1] while the remaining 10% represents rare malignancies (unusual forms of squamous cell carcinoma, minor salivary gland tumors, melanomas, lymphomas, sarcomas), and a variety of malignant tumors of odontogenic origin. Lifestyle, habits, and demographic, as well as genetic factors, influence geographic variations in the incidence of disease. In North America, common risk factors for the development of cancer of the

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oral cavity include tobacco and alcohol use. Outside of North America, dietary habits like chewing beetle, areca nut, and tobacco, represent additional risks for the development of oral cancer. Beyond these risks, there is little evidence linking dietary factors or nutritional deficiencies to the development of oral cavity cancer especially low fruit and vegetable consumption and high fat and/or sugar intake. The highest rates of incidence of cancer of oral cavity are observed in Pakistan, Brazil, India, and France [2]. While the use of alcohol and tobacco independently represent risk factors for the development of oral cavity cancer, the synergistic effect of these risk factors has been well documented. It has been suggested that the use of alcohol suppresses DNA repair following exposure to nitrosamine compounds; however, the exact mechanism of the observed synergy remains poorly defined. Human papillomavirus (HPV) is strongly associated with the development of oropharyngeal cancer and a small percentage of oral cavity cancers [3]. Over the past 30 years, the proportion of potentially HPV-related oral cancer in the USA has increased, possibly due to changing sexual behaviors especially in the young population. This probably explains the increasing number of patients with oral carcinoma who had never been exposed in tobacco or alcohol. During the last 30 years there has been an explosion of accumulated knowledge and evidence in our understanding of the biological phenomenon of oral carcinogenesis as well as in the technological advances in the diagnosis of the disease in both the histopathological and clinical levels. An equal abundance of knowledge has been achieved in the therapeutic management of the disease from the combined uses of surgery, radiotherapy, and chemotherapy. Despite all these developments the 5 year overall survival of the disease has remained in the range of 50–60%. The quality of life though of the patients, which has become a major issue, has undoubtedly improved during these 30 years [4].

Principles of Oral Cavity Cancer Management The treatment of primary tumors from different head and neck subsites often overlaps. Treatment for oral cavity cancer in general, is highly complex, not only because of the variety of tumor subsites, but also because of the anatomic constraints of the head and neck region, and the importance of maintaining organ function after treatment. The factors that influence the choice of initial treatment are those related to the characteristics of the primary tumor, those related to the patient and those related to the therapeutic team (Tables  27.1–27.6) [5]. They are therefore classified under tumor, patient, and treatment factors. In the selection of optimal therapy for oral carcinoma, one should consider these three sets of parameters in primary treatment

A.D. Rapidis

planning. The ultimate goal of treatment of cancer of the oral cavity is to eradicate disease, preserve or restore form and function, minimize the sequelae of treatment and finally prevent the development of any subsequent new primary cancers. Table 27.1  Staging for Tumors of the Lip and Oral Cavity (According to Patel and Shah [5]) T (primary tumor size) TX Primary tumor cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ T1 Tumor 2 cm or less in greatest dimension T2 Tumor more than 2 cm but not more than 4 cm in greatest dimension T3 Tumor more than 4 cm in greatest dimension T4a Lip Tumor invades through cortical bone, inferior alveolar nerve, floor of mouth, or skin of face (i.e., chin or nose)a Oral Cavity Tumor invades through cortical bone, into deep extrinsic muscle of tongue (genioglossus, hyoglossus, palatoglossus, and styloglossus), maxillary sinus, or skin of face T4b Tumor involves masticator space, pterygoid plates, or skull base and/or encases internal carotid artery a Superficial erosion alone of bone/tooth socket by gingival primary is not sufficient to classify as T4

Table 27.2  Staging for tumors of the nasal cavity and paranasal sinuses (according to Patel and Shah [5]) T (primary tumor size) TX Primary tumor cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ Maxillary sinus T1 Tumor limited to the maxillary sinus mucosa with no erosion or destruction of bone T2 Tumor causing bone erosion or destruction including extension into the hard palate and/or middle nasal meatus, except extension to posterior wall of maxillary sinus, subcutaneous tissues, floor or medial wall of orbit, pterygoid fossa, ethmoid sinuses T3 Tumor invades any of the following: bone of the posterior wall of maxillary sinus, subcutaneous tissues, floor or medial wall of orbit, pterygoid fossa, ethmoid sinuses T4a Tumor invades anterior orbital contents, skin of cheek, pterygoid plates, infratemporal fossa, cribriform plate, sphenoid, or frontal sinuses T4b Tumor invades any of the following: orbital apex, dura, brain, middle cranial fossa, cranial nerves other than maxillary division of trigeminal nerve V2, nasopharynx, or clivus Nasal cavity and ethmoid sinus T1 Tumor restricted to any one subsite, with or without bony invasion T2 Tumor invading two subsites in a single region or extending to involve an adjacent region within the nasoethmoidal complex, with or without bony invasion T3 Tumor extends to invade the medial wall or floor of the orbit, maxillary sinus, palate, or cribriform plate T4a Tumor invades any of the following: anterior orbital contents, skin of nose or cheek, minimal extension to anterior cranial fossa, pterygoid plates, sphenoid, or frontal sinuses T4b Tumor invades any of the following: orbital apex, brain, middle cranial fossa, cranial nerves other than V2, nasopharynx, or clivus

27  Multidisciplinary Management of Oral Cavity and Maxillary Sinus Cancers Table 27.3  Staging for all head and neck sites except the nasopharynx and thyroid (according to Patel and Shah [5]) N (regional nodal status) Nx Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastasis in a single ipsilateral lymph node, 3 cm or less in greatest dimension N2 Metastasis in a single ipsilateral lymph node, more than 3 cm but not more than 6 cm in greatest dimension; or in multiple ipsilateral lymph nodes, none more than 6 cm in greatest dimension; or in bilateral or contralateral lymph nodes, none more than 6 cm in greatest dimension N2a Metastasis in a single ipsilateral lymph node more than 3 cm but not more than 6 cm in greatest dimension N2b Metastasis in multiple ipsilateral lymph nodes, none more than 6 cm in greatest dimension N2c Metastasis in bilateral or contralateral lymph nodes, none more than 6 cm in greatest dimension N3 Metastasis in a lymph more than 6 cm in greatest dimension

Table 27.4  Staging for head and neck tumors (according to Patel and Shah [5]) M (distant metastasis) Mx Distant metastasis cannot be assessed M0 No distant metastasis M1 Distant metastasis

Table  27.5  Stage grouping for all head and neck sites except the nasopharynx and thyroid (according to Patel and Shah [5]) Stage group T stage N stage M stage 0 Tis N0 M0 I T1 N0 M0 II T2 N0 M0 III T3 N0 M0 T1 N1 M0 T2 N1 M0 T3 N1 M0 IVA T4a N0 M0 T4a N1 M0 T1 N2 M0 T2 N2 M0 T3 N2 M0 T4a N2 M0 IVB T4b Any N M0 Any T N3 M0 IVC Any T Any N M1

Table 27.6  Algorithm of stage status in cancer of the oral cavity Staging of oral cavity cancer N1 N2 N3 N0 Stage 0 T1S Stage I T1 T2 Stage II T3 Stage III T4a Stage IVA T4b Stage IVB Stage IVC any T any N when M1

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The tumor factors that affect the choice of initial treatment of oral cancer represent the clinical and histopathological characteristics of the tumor and more specifically, the anatomical site, size (T Stage), location (anterior versus posterior), proximity to bone (mandible or maxilla), status of regional cervical lymph nodes, previous treatment, and histology (type, grade, and depth of invasion). The ability of the patient to tolerate an optimal therapeutic scheme is similarly an important factor influencing the choice of initial treatment. The patient’s acceptance of and compliance with the proposed treatment are similarly important considerations in designing an optimal treatment strategy. Additionally, the performance status, the previous medical history and the presence of additional comorbidities should also be taken in consideration. The factors related to the therapeutic team are also important and are related with the experience, dexterity, ability, and availability of technical support of the surgical team and its environment. Expertise in various disciplines including surgery, radiotherapy, chemotherapy, rehabilitation services, dental, and psycho-social support are all crucial in bringing about a successful outcome of the therapeutic program. For the purpose of providing an overview of treatment strategies in oral cancer patients, it is mandatory to group the oral squamous cell cancers into: Early-stage disease (stage I and II; no apparent lymph node involvement); and advanced disease which includes cancer metastatic to cervical lymph nodes (regionally advanced) and locally advanced primary tumors (stages T3 and T4).

Early-Stage Disease Approximately 30–40% of patients with oral cavity cancer present with early (stage I and II) disease. In general, these patients are treated with curative intent using either Surgery or Radiotherapy (RT). Because both modalities result in similar rates of local control and survival, the choice is usually based upon an assessment of competing morbidities, functional outcomes, and accessibility. One advantage of RT over surgery is the ability to electively encompass areas at high risk for subclinical involvement (i.e., cervical lymph nodes). Prophylactic treatment of the clinically negative neck (i.e., no evidence of pathologic lymphadenopathy either by clinical examination or radiographic study) is somewhat controversial. However, in general, prophylactic neck irradiation or lymph node dissection is recommended if the likelihood of neck recurrence at a specific site exceeds 15 %. Generally in tongue cancer the incidence of nodal metastasis depends upon the stage of the tumor. T1, T2, and T3 tongue cancers are associated with 30, 50, and 70% respective incidence of microscopic nodal metastasis. Selective neck dissection can be used to effectively treat clinically positive nodal disease in selected patients [6, 7].

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As surgical cures can often be achieved rapidly and with minimal morbidity, surgery has become the gold standard for management of early cancers of the oral cavity. Tumors involving the oral tongue can usually be managed through a transoral approach. While radiotherapy is equally effective for the treatment of early disease, the rates of long-term sequelae including xerostomia, dysphagia, and osteoradionecrosis are unacceptably high. Other advantages of surgery include the duration of treatment. Surgical therapy requires a single intervention while RT requires daily therapy over a period of several weeks in addition to possible catheter implants and the use of chemotherapy. Therefore, in resectable patients RT is usually reserved for those patients who are unable to undergo surgery [8].

Advanced-Stage Disease Advanced disease (Stage III and IV) of the oral cavity is best managed with multimodality therapy. Surgery coupled with preoperative or postoperative RT is often utilized for advanced disease. Although preoperative radiation has been proposed to decrease the tumor mass and therefore increase the “resectability” of the tumor, it is common practice to surgically resect the tumor based on the pre-radiation margins because islands of viable tumor may persist in the initial peripheral margins. Additionally, preoperative radiation is associated with a higher rate of postoperative complications. For these reasons, most centers perform surgery followed by postoperative radiation [9, 10].

The Role of Radiotherapy and Chemoradiotherapy as Treatment Modalities in Oral Cancer The current standard technique for delivery of radiotherapy (RT) to tumors involving the oral cavity is three-dimensional conformal RT (3D-cRT). As opposed to the historically twodimensional planning which relied on simulation X-ray films, treatment planning with 3D-cRT is based upon threedimensional information that is obtained on simulation CT scans. The radiation dose distribution is shown in three dimensions and doses to the treatment target as well as various organs are more accurately calculated. Modification of beam properties can be performed if needed to produce a conformal dose distribution to the treatment target [11]. Although primary surgical management has been advocated for advanced (T4) oral cavity cancers, recent evidence suggested that primary chemoradiotherapy (CRT) may be an

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effective treatment approach for selected patients with T4 lesions, with comparable rates of locoregional control, survival, and complications associated with primary surgical management and postoperative RT [12]. Over the last few years, intensity-modulated RT (IMRT) has been implemented in most radiation oncology centers and is becoming a dominant treatment technique for head and neck cancer (HNC). With the assistance of advanced computer technology, IMRT is capable of delivering radiation doses that are highly conformal to the target, with rapid dose falloff outside of target volumes. This technique permits high doses of RT to be delivered to tumors which lie in close proximity to critical normal organs [13]. The newest technology, image-guided radiation therapy (IGRT) is being introduced into radiation therapy practice. A CT scanner is incorporated into the linear accelerator, allowing target position verification in the treatment position. The capacity for near real-time imaging during treatment permits tumors to be treated with greater precision and accuracy than is possible with conventional IMRT, further reducing toxicity to normal tissues. For conventional fractionation RT, the dose for all gross disease (primary and nodal) is 70 to 72 Gy in 2 Gy fractions over 7 weeks. Subclinical regions of the neck are electively treated to 50 Gy in 25 fractions, while nodal regions with adjacent gross disease may receive 60 Gy in 30 fractions. IMRT also allows for the delivery of smaller radiation doses to the major salivary glands, thus reducing the risk of permanent post irradiation xerostomia. Most oral cavity tumors as with the majority of head and neck cancer, typically present with advanced-stage locoregional disease (stages III or IV) for which local and regional control with surgery and/or radiation has been the mainstay treatment. After the publication of the trials on larynx preservation strategies in both Europe and the USA [14, 15], there was a rapid proliferation of non-site-specific trials to further investigate organ preservation protocols in the treatment of advanced head and neck squamous cell carcinoma. Over 70 divergent randomized trials compared traditional locoregional treatments of surgery and radiation vs. locoregional treatment plus chemotherapy. Unfortunately, this enthusiasm was plagued by small sample sizes and a lack of statistical power to confidently detect even modest effects on survival, leading to mixed results and an obscured clinical picture [16–18]. Concomitant CRT may represent an acceptable alternative in selected advanced stage of oral cancer patients. In addition to the optimal combination of drugs, the role of altered fractionation RT schedules are also under active study [19]. Two main strategies of altered fractionation have been explored in order to increase the effective dose of RT delivered without magnifying toxicity. Hyperfractionation that delivers smaller doses of RT twice daily (1.1 to 1.2 Gy fractions

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compared to conventional daily 1.8 to 2.0 Gy fractions), allowing higher doses of RT to be administered (thereby improving local control) without a significantly higher risk of late complications [20]. Because delayed long-term toxicity of normal tissues is dependent on the size of the individual fractional dose, decreasing the size of each radiation fraction should permit utilization of a higher total doses without increasing late morbidity [21]. In practice, multiple daily treatments with smaller-than conventional fraction sizes are given over approximately the same treatment duration. Typically 1.1 to 1.2 Gy/fraction, two fractions per day, to total doses of 74 to 80 Gy have been employed. Accelerated fractionation RT schedules deliver the total dose of RT in shorter treatment duration. This seems to reduce the rapid tumor repopulation that is thought to occur during treatment interruptions [20]. A benefit for hyperfractionated compared to conventional fractionation RT in patients with locally advanced HNC has been shown in at least three prospective, randomized trials [21–23], and in meta-analyses of these trial data [14, 20]. Even in the absence of chemotherapy, significantly higher local control rates have been documented with both strategies compared to conventional fractionation RT alone, although demonstrating a survival benefit from either approach has been more difficult [24]. Taken together, these data support the view that accelerated treatments using splitcourse RT schedules or reduced total doses do not improve locoregional tumor control or overall survival. Accelerated treatments that employ continuous (rather than split-course) RT schedules, without compromising the total dose, improve local control. [21]. However, whether the added mucosal toxicity is justified by meaningful gains in survival remains an open question. Altered fractionation RT is considered by some to represent a standard approach for patients who are receiving RT alone as definitive treatment for oral cancer. However, it is important to clarify that the indications for postoperative RT directed to the primary site are different from the indications for postoperative radiation directed at the neck. The goal of a surgical excision is to achieve a complete resection of the tumor with tumor-free margins. In cases where there are positive or close margins (tumor within 5 mm of the surgical margin), surgical re-resection is usually recommended. In cases where a re-resection is performed, if there remains evidence of microscopically positive margins, radiation directed at the primary site should be considered. In cases where there is neck disease that is N2 or greater, or the histopathological characteristics of the primary tumor demonstrate an aggressive behavior [25], radiation therapy to the neck is warranted, usually administered with concurrent chemotherapy [26, 27]. Definitive RT, usually administered with cisplatin-based chemotherapy, is the treatment of choice for patients with potentially resectable locoregionally advanced oral cancer

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who desire organ preservation, for those who have surgically unresectable disease, or who are medically inoperable. Although direct comparative data are lacking, combined chemotherapy and RT appears to produce similar locoregional control and survival rates as does surgery, while providing the opportunity for function preservation [24]. Chemotherapy can be administrated before, at the same time or after locoregional treatment corresponding to induction, concomitant, or adjuvant chemotherapy. There are several other potential advantages to giving neoadjuvant rather than postoperative (adjuvant) chemotherapy. These include the delivery of chemotherapeutic drugs through an intact vasculature which is optimal to enhance its therapeutic effectiveness before surgery or radiation. The neoadjuvant treatment is more likely to treat micrometastases, thus diminishing the chances of developing gross metastatic disease. Finally, the reduction in tumor size and healing prior to definitive RT may improve functional outcomes. The response to chemotherapy may be an important predictor of survival, as various studies have shown that patients with a good response to induction chemotherapy have a better overall survival [4, 28, 29]. A greater benefit (8%) was observed in trials that gave CT concomitantly to RT. Effect of concomitant CT on survival did not differ significantly between the group of trials with postoperative RT, or curative RT with conventional or altered fractionation. No significant difference was also seen between mono- and poly-chemotherapy. In the polychemotherapy group, the effect of chemotherapy was not significantly different between the different subgroups: with cisplatin or carboplatin (platin) and 5-fluorouracil (5-FU), with either platin or 5-FU or with neither [30, 31]. As might be expected, the proportion of deaths not due to head and neck cancer increases progressively with age from 15% in patients less than 50 to an impressive 39% in patients 71 and over. The survival benefit resulting from the addition of CT to RT is confirmed to be around 4%. This benefit is larger for concomitant CT, whereas there was no clear evidence of a benefit for induction and adjuvant CTs. Another important issue is that the benefit of concomitant CT appears to be similar irrespective of whether the RT is given conventionally or using altered fractionation. Finally, the magnitude of the benefit of concomitant CT is less in older patients, a feature that has also been observed with altered fractionation compared to conventional RT in head and neck cancer [20] and also when combining anti-EGFR agents (cetuximab) with radiotherapy [32–34]. In a recent meta-analysis the comparison of the benefit associated with concomitant vs. induction CT was examined. It is interesting that both the indirect and the direct comparisons were consistent on survival, event-free survival and locoregional failure, showing a clear advantage in favor of concomitant CT [35, 36].

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Postoperative RT with or without concomitant chemotherapy is reserved for those cases in which the risk of recurrence is high. Defining the “high-risk” patient has been the topic of controversy. This decision is made after a careful evaluation of the various patient and disease factors. The findings can be summarized as follows: Extracapsular extension and/or microscopically involved surgical margins are the only risk factors for which the impact on survival of adding chemotherapy to RT is statistically significant. There is a trend toward improved survival in favor of CRT in patients who had stage III–IV disease, perineural infiltration, vascular embolisms, and/or clinically enlarged levels IV–V lymph nodes secondary to tumors arising in the oral cavity or oropharynx. The differences though were not statistically significant. Patients with two or more histopathologically involved lymph nodes without extracapsular extension did not seem to benefit from the addition of CT. The problem with CRT in head and neck cancer is that the schedules are often rather toxic and associated with a substantial morbidity which in turn influences the compliance with treatment. Obviously, this morbidity is to some extent outnumbered by the benefit of the combined treatment, resulting in an improved survival; but we must not forget that many patients do not comply with treatment, and patients who do not fulfill a planned course of RT due to morbidity with the interacting drug, are in fact in a worse situation condition than the ones who are treated with RT alone.

Site-Specific Treatment The anatomic boundaries of the oral cavity extend from the skin–vermilion junction of the lips to the junction of the hard and soft palate above, and to the line of circumvallate papilla of the tongue below. Specific sites of tumor origin include the lips, floor of the mouth, oral tongue, lower alveolar ridge and retromolar trigone, upper alveolar ridge and hard palate, and the buccal mucosa [37]. The maxillary sinus carcinomas will also be included.

Lip Cancer The lip is the most common primary site within the oral cavity, accounting for approximately 25% of cancers at this site. The majority of lesions occur on the lower lip and 95 % occur in males [38]. Basal cell carcinomas (BCCs) may arise from the skin and cross the vermilion border to invade the lip, while squamous cell cancers (SCCs) most frequently develop at the vermilion margin. BCCs are more common on the upper lip. The similar local control and cure rates that can

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be achieved with surgery or RT in stage I lower lip tumors make either treatment acceptable. Surgery is the treatment of choice for early-stage lesions and is preferred because of better cosmetic results and lower morbidity rates compared to RT. Defects that involve less than two-thirds of the lip usually can be closed primarily. Defects involving two-thirds of the lip can be reconstructed with full thickness pedicled flaps (“Abbe or Estlander”) from the upper or lower lip [39]. Many reconstructive options are available for defects larger than two-thirds of the lip, ranging from local nasolabial flaps to hair-bearing free flaps. The facial artery musculomucosal flap has shown application and success in upper and lower lip reconstruction [40]. Radiation therapy is generally reserved for recurrent tumors, nodal disease, and for patients who cannot tolerate surgery. Maximum tumor thickness is a predictor of metastatic spread to the regional nodes, and is therefore important for treatment planning and assessment of prognosis in patients with squamous cell carcinoma [41, 42]. Among patients who have a clinically negative neck, those with T2 or larger tumors that are treated surgically should undergo ipsilateral neck dissection [42]. Upper lip and commissure tumors are more aggressive, tend to grow more rapidly, ulcerate sooner and metastasize earlier than those of the lower lip. Carcinomas in these sites may give regional metastases to preauricular and submandibular nodes.

Oral Tongue Cancer The incidence of tongue cancer exceeds all other sites in the oral cavity, excluding lip cancer, accounting for almost 30% of oral cancer patients. The median age for patients with SCC of the tongue is 60 and, similar to other disease sites, the male to female ratio is 3:1. Cancers of the mobile tongue have a high incidence of occult and clinical cervical lymph node metastases. Tongue cancer has been considered to have a more aggressive course in younger patients. However, more recent studies have found no difference in staging or survival among patients under the age of 40 as compared to a group of patients aged 60 to 70 [43, 44]. Those receiving neck dissection for prognostic or therapeutic purposes have significantly better 5-year survival rates than those who do not receive a neck dissection as part of their primary treatment. Surgery is recommended for small, anterior, and well-lateralized lesions. Radiation therapy is preferred for large T1 lesions and for T2 tumors where resection would result in impairment of normal speech and/or swallowing (Fig. 27.1). Most stage I and II lesions can be resected via an intraoral approach with ample surgical margins. Due to the small size

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Fig. 27.1  Squamous cell carcinoma of the tongue in a 48-years old female patient. The MRI shows the lesion extending and occupying the right side of the tongue. T1 weighting (a) and T2 weighting (b)

of these early tumors in relationship to the usual bulky mass of the tongue, most T1 and T2 cancers of the oral tongue can be excised without permanent speech or swallowing deficits. Excision usually entails a partial glossectomy. Adequate margins (>1 cm) and elective treatment of the clinically negative neck are extremely important in the treatment of early tongue cancer. The 5-year survival rate, in patients with stage I or II disease, after appropriate surgical treatment, approaches 90%. Elective neck dissection is recommended in patients with T2-4 tumors and a clinically negative neck because of the high incidence of occult cervical nodal disease [45–47]. More than 25% of patients undergoing elective neck dissection will be found with pathologically node positive (N+) [46]. The staging information provided by the neck dissection is crucial for defining necessity for and type of postoperative additional treatment. It is more difficult to define the role of elective neck dissection in patients with T1 disease and a clinically negative neck. There are no randomized trials examining this issue. The 5-year survival rates for patients undergoing synchronous (prophylactic) neck dissection, no dissection, or a metachronous dissection (at the time of clinical neck recurrence) are 81, 60, and 45%, respectively. This finding supports the concept that prophylactic neck dissections offer improved survival compared to the “wait and see” policy, and emphasizes the need for a more aggressive approach to the neck at primary tumor presentation [48]. The best pathologic predictors for the presence of occult neck metastases are depth of invasion above 5 mm, depth of muscle invasion, double DNA aneuploidy, and poor histologic differentiation.

It is therefore recommended that elective neck dissection must be considered in patients with T1N0 cancer undergoing surgical treatment of the primary who have aneuploid tumors, depth of muscle invasion >4 mm, or a poorly differentiated cancer [48]. As oral cavity cancer rarely metastasizes to neck level V, a radical or modified radical neck dissection of all five nodal levels is not necessary for patients with N0 neck. Selective neck dissection of levels I–III (“supraomohyoid neck dissection”) is the procedure of choice for elective neck dissection of the neck. Most of the relatively small numbers of isolated metastasis to level IV are from primary tumors of the tongue, which are known to produce “skip metastases.” Thus an “extended supraomohyoid neck dissection” of levels I–IV is recommended for elective treatment of the neck in tongue cancer in patients with T2 and above and N0 necks [49]. A number of recent prospective multi-institutional studies have demonstrated that sublevel IIB is rarely involved with isolated metastasis from oral cavity primary tumors, except from some tongue cancers [50–54]. Thus, it is justifiable to omit dissection of sublevel IIB in elective treatment of most cases of oral cavity cancers. In this way injury to the spinal accessory nerve is avoided [55]. It is recommended that elective neck dissection is performed for all patients with T2 or larger tumors if surgery is used to treat the primary tumor [47]. Ipsilateral neck dissection is generally sufficient for most T1/T2 tumors. However, bilateral node dissection should be considered for patients with anterior or midline lesions, as well as for those with more advanced stage disease (Fig. 27.2).

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Fig. 27.2  Squamous cell carcinoma of the tongue in a 55-years old male patient. The CT shows the lesion occupying the entire musculature of the left side of the tongue. Regional node metastases are also present: (a) at the level of the floor of the mouth; (b) AT the level of the base of the tongue. Multiple nodal metastases with central necrosis can be seen; (c) at the level of the hyoid bone. A large nodal block can be seen under the sternocleidomastoid muscle

Floor of Mouth Cancer The floor of the mouth is rich in neural and vascular structures including the lingual and hypoglossal nerves, the submandibular duct, and the sublingual glands. SCC of the floor of the mouth are aggressive oral cavity neoplasms. They typically present as painful infiltrative ulcerative lesions that may bleed. The lack of any substantial fascial barrier means that early tumors of the floor of mouth can quickly invade in to the underlying structures and metastasize to the first echelon lymph node basin (neck levels I and II). They have a high incidence of cervical nodal metastases which are detectable clinically in 30–60% of patients at presentation. The incidence of occult cervical metastases is also high [56]. Treatment approaches include surgery and RT. Due to the risk of radiation-induced bone necrosis, surgery is usually the preferred treatment approach in operable patients. Local control of these tumors can be difficult because of their proximity to the mandible and the lack of a good mechanical barrier to

tumor spread at this site. Surgery is generally preferred with an emphasis on negative margins, which can be technically difficult without rim mandibulectomy due to the proximity of and/or occult invasion into the mandible. The outcome of surgical treatment for patients with cancer of the floor of the mouth varies directly with tumor size and the status of the surgical margins. In early-stage T1 and T2 disease, the 5-year survival can be higher than 80% [56, 57]. Due to the high incidence of occult nodal disease in all but the earliest superficial carcinomas (i.e., those limited to less than 5  mm invasion) of the floor of the mouth, prophylactic neck dissection is recommended at these sites [45, 56]. For T1 or T2 lesions, an ipsilateral supraomohyoid (level I to III), dissection is generally advocated as the surgical procedure of choice; bilateral selective dissections are indicated for more anterior/midline lesions [58]. Because of the density of neurovascular structures in the floor of the mouth, frequent metastasis occurs to the sublingual, submandibular, and level II lymph node basins.

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Postoperative radiation (in some cases, with concomitant chemotherapy) is indicated for patients who have positive resection margins (if not re-resected), mandibular bone erosion, or pathologically positive lymph nodes after elective neck dissection. Postoperative RT should also be considered if there is vascular or perineural invasion in the primary tumor [59]. For resectable tumors in nonsurgical candidates, RT (usually a combination of external beam RT and brachytherapy) achieves similar local control rates [59].

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Tumors within the oral cavity may invade the mandible and gain entrance into the mandibular canal through several routes. Not uncommonly, SCC of the oral epithelium will travel along the surface mucosa until it approaches the attached gingiva where the tumor cells may come into contact with the periosteum of the mandible. This can be done in both dentate and edentulous patients. In the dentate patient tumor cells demonstrate a tendency to migrate into the dental sockets because this area represents a pathway of minimal

resistance. In edentulous patient, tumor cells will migrate onto the occlusal surface of the alveolus and enter the mandible through dental pits, which are cortical bone defects at the location of prior dentition. SCCs of the floor of the mouth may also extend to invade the neighboring mandibular bone. Less commonly, tumor may enter the mandible through mental or mandibular canals. Finally, adjacent tumor may erode through the cortical bone directly into the mandibular canal. Plain radiography has been used in the past for the diagnosis of tumor invasion of the mandible. The introduction of orthopantomogram or panoramic radiography, CT, and MRI scans have increased the accuracy of preoperative imaging and staging (Fig. 27.3). Significant debate still exists regarding the optimal modality or combination of modalities recommended for preoperative assessment of mandibular invasion by oral SCC. While CT is a very accurate method for identifying gross bone invasion, prior work has suggested that bone invasion may be missed in as many as 27% of patients with preoperative CT scans [60]. The CT scan renders an excellent view of both the soft tissue and bone of the mandible; however, it has several limitations, the most significant being artifacts caused by dental amalgams and prosthetic metal bridgework. Dental amalgams commonly create

Fig. 27.3  Squamous cell carcinoma of the anterior part of the mandible in a 60-years old female (a). Orthopantomogram showing the lesion to extent from the right premolars area of the mandible to the left one (arrows) (b). CT of the mandible shows the extensive distraction of the

osseous architecture of the mandible extending to the buccal and lingual cortical bone (c–e). Bone Scan with Tc 99 m shows a pathological uptake of the radionuclide in the anterior part of the mandible. The uptake corresponds to the extend of the lesion

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a shadow leading to artifact that can obscure invasion of the mandibular cortex. Additionally, the CT scan may misleadingly detect defects in the cortex secondary to irregular tooth sockets or periapical lesions of inflammatory origin. In light of these shortcomings, several investigators have reported on the use of a Dentascan. The Dentascan was introduced in the early 1980s to assist oral maxillofacial surgeons in planning for osseointegrated implants. The Dentascan images are derived by reformatting standard axial CT scans in two views, panelliptical and parasagittal. This reformatting permits assessment of the buccal and lingual cortices. The diagnostic accuracy of the Dentascan is high, yielding a sensitivity of 95% and a specificity of 79% with a positive predictive value of 87% and a negative predictive value of 92% [61]. The Dentascan is therefore an accurate method for preoperative evaluation of mandibular invasion in patients with SCC of the oral cavity (Fig. 27.4). While the CT scan and Dentascan may offer excellent methods for assessing bone, MRI offers the advantage of imaging soft tissue and potentially the medullary bone space. Several studies have examined the use of MRI in assessing mandibular invasion and it has been concluded that MRI is superior for evaluating the medullary space of the mandible [62] but inadequate for assessing mandibular invasion. Shaha [63] examined the value of various studies including panoramic X-rays, dental films, routine mandible films, bone scans, CT scans, and MRI and found that CT scanning was

not very helpful mainly because of the presence of irregular dental sockets and artifacts. Many suggest that clinical evaluation is the most accurate in determining the presence of bone invasion and the optimal method of resection, marginal vs. segmental [64]. Most centers consider the combination of a CT scan and a panoramic X-ray acceptable for preoperative imaging of the mandible and maxilla; however, the most accurate measure of bony invasion is determined clinically at the time of surgery. Unless there is frank invasion of the bony cortex, periosteal stripping followed by frozen section examination at the time of surgery is often the most reliable measure of bone invasion. Recent studies have shown that technetium (Tc) 99m bone scintigraphy in the form of planar views or as SPECT provide a high diagnostic accuracy for mandibular invasion by oral SCC of the alveolous in both edentulous and dentate patients [65, 66]. Among all investigations and evaluations of the extent of disease in the oral cavity in relation to involvement of the mandible, the best investigation continues to be routine clinical evaluation and intraoperative evaluation of the proximity of the tumor to the inner border of the mandible. Even though the tumor may not involve the mandible directly, a marginal mandibulectomy may be necessary for appropriate oncologic margins and resection of part of the mandible due to close proximity. This decision is best made using clinical judgment.

Fig. 27.4  Squamous cell carcinoma of the left body of the mandible in a 68-years old male patient (a). The orthopantomogram shows a lytic lesion in the left body of the mandible extending to the inferior dental canal (b). The CT shows complete destruction of the entire width of the

mandibular body (c). The Denta Scan CT depicts the erosion of the cortical bone and the extension of the tumor to the medullary part of the mandible (d). Three-dimensional reconstruction of the CT of the mandible

27  Multidisciplinary Management of Oral Cavity and Maxillary Sinus Cancers

Tumors invading the mandible can be managed either with a marginal resection or a segmental resection. The decision regarding the optimal method of tumor resection is largely dependent on the degree of invasion. It has been suggested that tumor invasion of the periosteum or cortical bone, without invasion of the medullary cortex, can be appropriately managed with a marginal resection. Tumors that erode into the medullary canal, however, require a segmental resection. It has been shown that once a tumor gains access to the medullary canal, tumor may travel through the canal via the neurovascular bundle. The inability to obtain frozen section assessment of the mandible intraoperatively represents a management dilemma because decalcification of the mandible specimen in preparation for definitive histopathological analysis can take as long as 2 weeks. The periosteum is relatively resistant to cancer invasion. With the exception of the tooth sockets, the periosteum acts as a dense barrier to the invasion of adjacent tumor. In spite of the protective periosteum, aggressive and longstanding tumors erode the periosteum and invade the adjacent mandible through a variety of pathways. Two distinct histological patterns of tumor invasion have been identified. The first pattern is referred to as infiltrative and is characterized by finger-like projections of tumor which advance independently and invade the cancellous spaces without the intervening connective tissue layer and possess very little osteoclastic activity. The second pattern is referred to as erosive. In contrast to the infiltrative pattern, the erosive pattern is characterized by a broad front with a connective tissue layer and active osteoclast activity. The significance of the erosive and infiltrative patterns has been demonstrated in several reports, and it has been demonstrated that patient survival is significantly impacted by the pattern of invasion [67]. It has been suggested that the pattern of invasion is a reflection of the biologic aggressiveness of the tumor and may impact the approach to ablative therapy. While most tumors that invade the mandible mandate postoperative external beam radiation, some have suggested that superficially invading tumors may not benefit from postoperative radiation. Given the aggressive behavior of the infiltrative pattern of invasion, we recommend postoperative RT for all patients with this pattern of bone invasion. While the superficial invasion of the periosteum or cortical bone may be managed with a marginal mandibulectomy, once the tumor has eroded into the medullary cavity and mandibular canal most advocate a segmental resection. Determining the presence of bone erosion and the extent of bone erosion represents an ongoing clinical dilemma. The poor predictability associated with preoperative imaging has led many to rely on preoperative clinical assessment as the primary method for determining the presence of mandibular invasion. Several groups have studied this issue and found that clinical evaluation of mandibular bone erosion is more

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sensitive than radiographic evaluation; however, radiographic assessment may be more specific and provide a higher reliability index [68]. There are a few studies reviewing the impact of clinical assessment alone in determining the extent of mandibular invasion. This likely represents the difficulty in quantifying a clinical exam. However, most agree that clinical assessment for invasion is paramount. Several studies have evaluated the role of periosteal stripping as an indicator for tumor invasion of the mandible and found that periosteal stripping at the time of resection represented an accurate predictor of the presence of mandibular invasion [69]. Without clear preoperative evidence of mandibular invasion, a marginal resection followed by periosteal stripping and inspection is an adequate approach. In the event that microscopic evidence of invasion at the rim is discovered, the marginal mandibulectomy is converted into a segmental mandibulectomy.

Lower Alveolar Ridge and Retromolar Trigone Cancer The retromolar trigone is a small mucosal space that begins at the third molar of the mandible and extends cranially to  the maxillary tuberosity. It is directly continuous with the buccal mucosa, upper and lower gingiva, maxillary ­tuberosity, anterior tonsillar pillar, soft palate, and the floor of the mouth. Squamous cell cancers arising in the retromolar trigone and lower alveolar ridge comprise approximately 10% of all oral cancers and exhibit the same 3 or 4:1 male predominance of other head and neck cancers. The presenting symptom is typically pain, which is exacerbated by chewing. Treatment options include RT and surgery. The local recurrence rate is higher with these tumors than for other sites in the oral cavity due to microscopic extension to the mandible and maxilla (for retromolar trigone tumors). In addition, the probability of occult regional lymph node metastases is higher than with most other oral cavity tumors, with the exception of tongue cancer and floor of mouth cancer [62]. Thus, elective neck dissection is usually recommended for patients with a clinically negative neck. Surgical treatment involves wide local excision. Marginal or horizontal “rim” mandibulectomy may be required in order to achieve tumor-free margins. Due to the normally thin overlying mucosa and the close proximity to the mandible, alveolar ridge and retromolar sites have a propensity for early invasion of this bone, as well as the maxilla for retromolar trigone lesions [70, 71]. Consequently, lesions that are clinically staged T1/T2 and treated with rim mandibulectomy may become pathologic stage T4 after histologic

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confirmation of bony invasion. Segmental or composite resection is reserved for those tumors that are deeply invasive or that wrap around the mandible [60]. In addition, segmental mandibulectomy may be necessary for early-stage lesions in the thin, edentulous mandible in order to achieve negative margins. It is extremely important to determine the true invasive margin, which may extend grossly or microscopically beyond the tumor front [62]. Determining this invasive margin is challenging. For oral cavity lesions in general, computed tomography (CT) scans may be helpful for identifying bone invasion. The sensitivity of CT scan for bone involvement of the retromolar trigone is approximately 50% with a negative predictive value of 60%; however, the positive predictive value is approximately 90%. It has been concluded that while the CT scan is accurate when bone erosion is clearly identified, its negative predictive value is unacceptably low and therefore an inaccurate indicator of bone invasion at the retromolar trigone. In one report of 127 patients with oral cavity or oropharyngeal carcinoma treated with composite (segmental) resections, CT scan findings suspicious for bone invasion and primary tumor location (alveolus, retromolar trigone, tonsil, and sulcus) were the only independent variables that predicted for the presence of bony invasion [65, 70, 72]. However, in one report, preoperative CT scan failed to identify bone invasion in one-half of retromolar trigone lesions that histologically invaded bone [73]. Potential reasons for this low sensitivity include the thickness of CT sections, the lack of bone windows and coronal imaging, and the presence of distortion from dental artifact. A resection margin of at least 1  cm in all directions is recommended [74]. At least for tumors involving the retromolar trigone, the optimal extent of surgery is controversial [56, 75]. In addition to stage, outcomes are dependent on the presence of bone invasion, deep infiltration of the masticator space, nodal involvement, and treatment modality [71, 76, 77]. Among the patients with stage I and II disease, survival exceeds 75% at 5 years. In a later series of 99 patients treated with definitive RT or surgery followed by RT, local control rates were better in surgically treated patients (approximately 71% vs. 48%) [76, 78]. Among all patients treated for stage I to III disease (RT or surgery plus RT), 5-year rates of causespecific and overall survival were 70 and 58%, compared to 57 and 42% for those treated for stage IV disease. Notably, in multivariate analysis, both cause-specific and overall survival were significantly better in the group undergoing RT in addition to surgery. For early lesions of the lower alveolar ridge and retromolar trigone, selective neck dissection in levels I–III is recommended as tumors are characterized by early invasion of the mandible and high rates of regional metastases.

A.D. Rapidis

Tumors Invading the Buccal Mucosa Buccal cancer comprises less than 10% of oral cavity cancers and when it occurs, it commonly arises from a preexisting leukoplakia [79, 80]. SCCs arising within the buccal mucosa are notable for their locoregional aggressiveness. For earlystage disease, treatment with either surgery or definitive RT is reasonable, although in most circumstances surgery is favored. Surgical treatment can be compromised by anatomic difficulties in obtaining adequate margins. For locally advanced but resectable tumors, surgery followed by postoperative RT is the treatment of choice. The principals of management of buccal cancer are no different than those of other subsites within the oral cavity. Surgical therapy is the preferred method of management. In early disease, surgical excision can usually be accomplished transorally. The buccal space has poor anatomic boundaries and it is difficult to obtain a clear surgical margin. Even patients with early-stage disease have potential microscopic invasion through the buccinator muscle into the buccal fat and buccal space. Although more aggressive surgery including exenteration of the buccal space and parotidectomy may improve surgical results, the resulting disfigurement and morbidity of these procedures may be considerable. Tumors that invade the buccinator muscle and tumors that present with nodal disease or with poor prognostic features should be managed with postoperative radiation therapy. Negative surgical margins are paramount and in an effort to achieve this goal, careful preoperative planning is essential to determine the extent of the tumor. While early tumors of the buccal mucosa commonly present as an irregular mucosal mass, more than half of buccal tumors will present as deeply invasive tumors that may track along the parotid duct, masseter muscle or into the palate. The proximity of the buccal mucosa to the parotid duct requires that the duct be traced retrograde and sampled to ensure a negative margin. Deeply invasive lesions may break into the buccal fat pad. When this occurs, it is advisable to resect the entire fat pad because negative surgical margins in this area are difficult to confirm. The rich lymphatic network, characteristic of the buccal region, and the high rate of lymph node metastasis, mandate that the neck be carefully evaluated and, in most cases, treated. Smaller tumors can usually be managed through a transoral approach; however, more advanced tumors may require a midline labiotomy incision. Cancer of the buccal mucosa is a highly aggressive form of oral cavity cancer that is associated with a high rate of locoregional recurrence and poor survival. Surgery is generally preferred for managing small lesions. The tumor can usually be excised using a transoral approach. Five-year survival rates are approximately 75% for patients with stage I disease and 65% for patients with stage II

27  Multidisciplinary Management of Oral Cavity and Maxillary Sinus Cancers

lesions [81–83]. However, local recurrence rates with surgery alone are high, particularly with surgical margins less than 2 mm [81, 82, 84]. Treatment of the clinically negative neck is controversial. Elective neck dissection is not routinely recommended in all patients. For those with small (T1) lesions, cervical lymph node metastases occur in less than 10% and the neck can be observed. Selective neck dissection of levels I to III should be considered for larger lesions [82].

Upper Alveolar Ridge and Hard Palate Cancer Malignant neoplasms of the upper alveolar ridge and hard palate comprise approximately 5% of oral cavity malignancies and have a male to female ratio of 8:1. Only about two-thirds of hard palate malignant neoplasms are SCCs; the remainders are minor salivary gland carcinomas and other rare malignancies. Unlike other areas of the oral cavity where SCC makes up the overwhelming majority of pathology, the palate is rich in minor salivary glands and therefore is the site of both benign and malignant salivary gland tumors. Most upper alveolar ridge and hard palate SCCs are managed with primary surgery. RT can be used for small, superficial lesions, or tumors with extensive involvement of the hard and/or soft palate. Combined modality therapy provides better locoregional disease control than single modality therapy [76, 77]. Postoperative RT (in some cases with concomitant chemotherapy) is indicated for patients with positive resection margins, bone erosion, or pathologically positive lymph nodes after elective neck dissection [76, 77]. Others recommend that postoperative RT also be considered if there is vascular or perineural invasion in the primary tumor [59]. The principals of management of tumors of the palate are similar to those of mandible; obtaining tumor-free margins is essential to achieving a good outcome. Lateral tumors may represent a risk to invasion and perineural spread via the palatine or trigeminal neurovascular bundle. The depth of invasion will dictate the extent of the surgical resection. Superficial lesions of the palatal mucosa are best managed with a wide surgical resection including the underlying palatal periosteum. The periosteum serves as an early barrier to spread; however, as tumors become more invasive, tumors can vertically invade the nasal vault or maxillary sinus. Tumors of the hard palate rarely metastasize to the neck and therefore a neck dissection is rarely warranted in the absence of demonstrable regional disease. One exception is when there is tumor erosion through the posterior or posterior lateral maxillary sinus into the pterygopalatine fossa. Most lesions of the upper alveolar ridge and hard palate are managed with primary surgery. Lesions with extensive involvement of the hard and/or soft palate can also be

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initially treated with primary RT. In patients initially treated with surgical resection, the 5-year survival rates are 70 and 45% for patients with stage I and II disease [85]. Selective neck dissection with removal of levels I–III nodal groups is adequate for early disease of the hard palate in patients with clinical positive nodes at presentation. If disease extends beyond the hard palate, however, elective treatment of the neck is indicated even in No neck patients.

Maxillary Sinus Cancer Paranasal sinus cancer is rare, accounting for just 3% of upper aerodigestive tract malignancies [86]. The incidence is higher in males than in females (2:1) with a peak incidence at 50–59 years of age. Lesions of the maxillary sinus are most common, followed by the ethmoid, sphenoid, and frontal sinuses. These tumors are generally slow-growing and tend to remain asymptomatic until late in the course. As a result, most patients present with locally advanced disease. SCCs constitute the majority of paranasal malignancies (45–80% of cases). This is followed by malignancies of salivary gland origin, of which adenoid cystic carcinomas predominate [87–89], followed by adenocarcinomas and mucoepidermoid carcinomas. The most common symptoms in patients with paranasal sinus cancer include facial or dental pain, nasal obstruction, and epistaxis [90]. Oral symptoms (e.g., ill-fitting dentures) occur in 25–30% of patients. Pain with unilateral nasal obstruction or ocular symptoms can be seen in 50 and 25% of patients with antral-ethmoidal disease, respectively. A classic triad of facial asymmetry, palpable/ visible tumor in the oral cavity, and visible intranasal tumor occurs in 40–60% of patients with advanced disease. At least one of these signs is present in 90% of cases [91]. As disease progresses, symptoms and signs depend upon the involved site. The bony structures between the nasal cavity, sinuses, orbits, and cranial vaults are thin and offer little resistance to cancer spread (Fig.  27.5). Regional nodal metastases are uncommon, occurring in less than 20% of patients, lower if they have adenoid cystic tumors [87, 92–95]. The incidence of lymph node involvement increases as tumors extend locally to adjacent sites, especially with extension into the oral cavity. The retropharyngeal nodes comprise the first echelon lymphatic drainage for sinus malignancies. Other regional nodes that may be involved with lymphatic spread are the periparotid and level Ib nodes There is no consensus as to optimal treatment for earlystage tumors. Traditionally, surgery has been the primary treatment modality for paranasal sinus cancers involving the maxillary or ethmoid sinuses. However, the limitations of surgery alone are obvious given the frequent presentation of advanced disease [96].

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A.D. Rapidis

Fig. 27.5  Squamous cell carcinoma of the right maxillary antrum extending in the homolateral orbital cavity, the anterior ethmoids and the nasal cavity in a 72-years old male (a). CT shows the lesion occupying the right maxillary sinus. The lesion is confined within the maxillary sinus cavity and does not erode the wings of the sphenoid bone (b). The lesion occupies the anterior ethmoids and erodes the thin lateral orbital wall (c). Coronal section showing the extension of the tumor into the right orbital cavity (d). In the MRI (coronal T1 weight imaging) the tumor extends to the entire right middle third of the face (e). Sagittal T1 weighting image showing the tumor eroding the right orbital floor and extending into the content of the orbital cavity (f, g). T1 and T2 weighting images of the tumor invading the anterior ethmoids

Both, surgical technique and the overall approach to management have evolved to incorporate into the decision making process the histology and tumor size as well as location in relation to the adjacent critical structures. In many cases of maxillary and ethmoid sinus SCC, for example, aggressive local therapy includes en bloc craniofacial resection with or without orbital exenteration, followed by reconstruction and adjuvant RT. RT may be used, particularly for T1 tumors of the ethmoid, sphenoid and frontal sinuses, with acceptable results

[97, 98]. However, in practice, RT is rarely used as the sole modality of treatment except for cancers of the frontal and sphenoid sinuses, which are unsuitable for en bloc surgical resection. Elective neck treatment is not generally recommended given the low incidence of cervical metastases, unless there is significant soft tissue or skin involvement. Regardless of the surgical margin status, adjuvant postoperative RT optimizes local control. However, even with aggressive surgery and adjuvant RT, the results of treatment for most paranasal

27  Multidisciplinary Management of Oral Cavity and Maxillary Sinus Cancers

sinus cancers is poor with local control rates from 50 to 60%, and 5-year survival rates ranging from 30 to 60% [97–105]. Preoperative RT has been explored as a means of making these lesions more amenable to surgical resection [92, 106]. However, given the inherent bias in these nonrandomized studies, it is unclear whether preoperative is superior to postoperative RT in enhancing local control and improving outcome. The use of postoperative RT and concomitant chemotherapy should be considered in patients with pathologically positive lymph nodes, particularly in cases with adverse prognostic factors such as multiple metastatic lymph nodes or any node with extracapsular spread.

Conclusions If one wants to summarize the most notable developments of the last 30 years in the therapeutic management of oral squamous cell carcinomas that have been incorporated into everyday clinical practice, he should definitely point out the following key issues. During the last 30 years the belief that oral cancer management is based on team work has been established. The functions of tumor boards and combined clinics is a common contemporary practice with an exceedingly large number of medical, surgical and other specialties being part of comprehensive, multidisciplinary therapeutic head and neck teams.

The Basic Treatment Modalities Remain Surgery, Radiotherapy, and Chemotherapy Basic surgical techniques have not changed dramatically over the last 30 years. Among the major changes are the variations in the surgical management of the neck of both clinically negative and clinically positive neck patients, as well as the management of the mandible especially in the early invasion of oral squamous cell carcinoma in the mandibular bone. The revolution in the surgical treatment of oral cancer is the introduction of reconstructive techniques with both pedicled locoregional flaps and free tissue transfer. These reconstructive techniques allowed for safer and wider resections with adequate disease-free margins and functional reconstruction of the created surgical defects. Contemporary radiotherapeutic treatment has very little similarities with that of the late 1970s. Modern technology with the institution of new forms of radiation and the application of sophisticated computerized methods have enhanced the therapeutic effectiveness of irradiation with an equal impor­tant reduction in the sparing in irradiation of normal surrounding

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tissues. This has led to an increased therapeutic dose in the tumorous bed and a decreased severity of radiation-induced injuries in the neighboring, unaffected by the disease, normal tissues. Alterations in the fractionations have also shown to produce better therapeutic results in selected cases. The era of methotrexate, the leading chemotherapeutic agent of the 1970s, was followed by the institution of platinumbased chemotherapies with or without the addition of 5 Fu. Adjuvant and neoadjuvant schemes coupled with pre- or postoperative radiotherapy started in the late 1980s and showed a distinct survival benefit over radiotherapy alone. This major breakthrough was followed by the institution of various and diverse chemoradiation regimes tested over a large time period for their survival benefits. The introduction of taxanes and the development of molecular targeted therapies during the last 5 years have revolutionized the concept of chemoradiation. Induction chemotherapy and chemoradiation coupled with epidermal growth factor receptor antagonists proved to have a survival benefit in patients with locally advanced or recurrent squamous cell carcinoma of the head and neck. Other biological agents against tumor angiogenesis or resulting in the restoration of cell apoptosis are being tested in various phase I or II trials with promising results.

• In the course of the next decade: oral cancer in nonsmoker non-drinkers will increase. • The differences in the ratios between males and females will tend to equalize. • Surgery will remain the prime modality in early (stage I and II) disease. • Molecular prognosticators will be used to determine optimal treatment. • Postoperative chemoradiation will remain the treatment of choice for “aggressive” early (stage I and II) disease. • Organ preservation treatments will prevail in advanced (stage III and IV) disease. • Surgery will remain the treatment of choice for locoregional salvage surgery. • The use of stem cells and biomechanical engineering will compliment reconstructive surgery.

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380 83. Strome SE, To W, Strawderman M, et al. Squamous cell carcinoma of the buccal mucosa. Otolaryngol Head Neck Surg. 1999;120:375–9. 84. Nair MK, Sankaranarayanan R, Padmanabhan TK. Evaluation of the role of radiotherapy in the management of carcinoma of the buccal mucosa. Cancer. 1988;61:1326–31. 85. Delclos L. Afterloading interstitial irradiation techniques In: Levit SH, Khan FM, Potish RA, editors. Technological basis of radiation therapy. Philadelphia: Lea & Febiger; 1992. 86. What Are The Key Statistics About Nasal Cavity and Paranasal Sinus Cancers? In: http://www.cancer.org/docroot/CRI/content/ CRI_2_4_1X_What_are_the_key_statistics_for_nasal_cavity_ and_paranasal_cancer.asp?sitearea; Accessed on March 25, 2008. 87. Blanco AI, Chao KS, Ozyigit G, et  al. Carcinoma of paranasal sinuses: long-term outcomes with radiotherapy. Int J Radiat Oncol Biol Phys. 2004;59:51–8. 88. Harbo G, Grau C, Bundgaard T, et al. Cancer of the nasal cavity and paranasal sinuses. A clinico-pathological study of 277 patients. Acta Oncol. 1997;36:45–50. 89. Chen AM, Daly ME, Bucci MK, et al. Carcinomas of the paranasal sinuses and nasal cavity treated with radiotherapy at a single institution over five decades: are we making improvement? Int J Radiat Oncol Biol Phys. 2007;69:141–7. 90. Jansen EP, Keus RB, Hilgers FJ, Haas RL, Tan IB, Bartelink H. Does the combination of radiotherapy and debulking surgery favor survival in paranasal sinus carcinoma? Int J Radiat Oncol Biol Phys. 2000;48:27–35. 91. Howard DJ, Lund VJ, Wei WI. Craniofacial resection for tumors of the nasal cavity and paranasal sinuses: a 25-year experience. Head Neck. 2006;28:867–73. 92. Vrionis FD, Kienstra MA, Rivera M, Padhya TA. Malignant tumors of the anterior skull base. Cancer Control. 2004;11:144–51. 93. Ganly I, Patel SG, Singh B, et al. Craniofacial resection for malignant paranasal sinus tumors: Report of an International Collaborative Study. Head Neck. 2005;27:575–84. 94. Porceddu S, Martin J, Shanker G, et  al. Paranasal sinus tumors: Peter MacCallum Cancer Institute experience. Head Neck. 2004;26:322–30.

A.D. Rapidis 95. Kellman RM, Marentette L. The transglabellar/subcranial approach to the anterior skull base: a review of 72 cases. Arch Otolaryngol Head Neck Surg. 2001;127:687–90. 96. Shah JP, Gil Z. Current concepts in management of oral cancer– surgery. Oral Oncol. 2009;45:394–401. 97. Parsons JT, Mendenhall WM, Mancuso AA, Cassisi NJ, Million RR. Malignant tumors of the nasal cavity and ethmoid and sphenoid sinuses. Int J Radiat Oncol Biol Phys. 1988;14:11–22. 98. Waldron JN, O'Sullivan B, Warde P, et al. Ethmoid sinus cancer: twenty-nine cases managed with primary radiation therapy. Int J Radiat Oncol Biol Phys. 1998;41:361–9. 99. Tiwari R, van der Wal J, van der Waal I, Snow G. Studies of the anatomy and pathology of the orbit in carcinoma of the maxillary sinus and their impact on preservation of the eye in maxillectomy. Head Neck. 1998;20:193–6. 100. Vedrine PO, Thariat J, Merrot O, et al. Primary cancer of the sphenoid sinus – a GETTEC study. Head Neck. 2009;31:388–97. 101. Katz TS, Mendenhall WM, Morris CG, Amdur RJ, Hinerman RW, Villaret DB. Malignant tumors of the nasal cavity and paranasal sinuses. Head Neck. 2002;24:821–9. 102. Parsons JT, Kimsey FC, Mendenhall WM, Million RR, Cassisi NJ, Stringer SP. Radiation therapy for sinus malignancies. Otolaryngol Clin North Am. 1995;28:1259–68. 103. Brizel DM, Light K, Zhou SM, Marks LB. Conformal radiation therapy treatment planning reduces the dose to the optic structures for patients with tumors of the paranasal sinuses. Radiother Oncol. 1999;51:215–8. 104. Hoppe BS, Nelson CJ, Gomez DR, et al. Unresectable carcinoma of the paranasal sinuses: outcomes and toxicities. Int J Radiat Oncol Biol Phys. 2008;72:763–9. 105. National Comprehensive Cancer Network (NCCN) guidelines available online at http://www.nccn.org/professionals/physician_ gls/f_guidelines.asp. In; Accessed May 12, 2009. 106. Hoppe BS, Stegman LD, Zelefsky MJ, et al. Treatment of nasal cavity and paranasal sinus cancer with modern radiotherapy techniques in the postoperative setting – the MSKCC experience. Int J Radiat Oncol Biol Phys. 2007;67:691–702.

Chapter 28

Management of Nasopharyngeal Carcinoma Anne W.M. Lee, Wai-Tong Ng, Tsz-Kok Yau, Stephen C.K. Law, and William I. Wei

Abstract  Nasopharyngeal carcinoma (NPC) is a distinctly radiosensitive and chemosensitive tumor. Best quality radiotherapy is demanded to build up the complex concave highdose zone for this critical location. Intensity-modulated (IMRT) technique is advocated, image guidance to ensure setup precision and adaptive re-planning if major deviations from intended dose distribution occur during the treatment course are useful improvements if resources allow. Stringent dose constraint to organs at risk should be attempted to minimize late toxicities. Addition of cisplatin-based concurrent-adjuvant chemotherapy is recommended for patients with stages III–IVB and high-risk stage IIB diseases. More contemporary series using IMRT together with extensive use of chemotherapy and acceleration reported very encouraging early results with locoregional control in excess of 90% at 2–4 years; the key remaining problem is distant failure. Further improvement of efficacy by changing chemotherapy sequence to induction-concurrent is being explored. The plasma level of Epstein–Barr Viral Deoxyribonucleic Acid is an additional tool for nonkeratinizing carcinoma for prognostication and monitoring disease progress. Integrated fluorodeoxyglucose positron emission tomography and computed tomography is useful for excluding distant metastases and posttreatment persistent/recurrent disease. Early detection of failure is critical for increasing the chance of salvage; aggressive treatment should be attempted as far as possible, long survival can be achieved for patients with limited failure or metastasis. Different salvage methods and reported results are summarized. Keywords  Nasopharyngeal carcinoma • Radiotherapy • Concurrent chemotherapy • Salvage treatment • Late toxicity Nasopharyngeal carcinoma (NPC), particularly the classical non keratinizing type, is different from other head and neck cancers for its distinctly skewed geographic and ethnic distriA.W.M. Lee (*) Clinical Oncology, Pamela Youde Nethersole Eastern Hospital, Chai Wan, Hong Kong, China e-mail: [email protected]

bution, association with Epstein–Barr virus (EBV), and aggressive natural behavior with especially high predilection for distant metastases. Because of its deep-seated location and anatomical proximity to critical structures, radical surgical resection is very difficult. The role of surgery is mainly biopsy for histological confirmation and salvage of persistent or recurrent disease. This cancer is highly radiosensitive and chemosensitive; but the therapeutic margin is notoriously narrow, thorough knowledge of its complex anatomy and natural behavior (Fig. 28.1) is crucial in managing this great challenge.

Investigations and Staging Evaluation of locoregional extent should include complete physical examination (particularly on involvement of cranial nerves and cervical nodes), endoscopy, and cross-sectional imaging. Magnetic resonance imaging (MRI) is preferred over computed tomography (CT) because of its superior sensitivity. A study by Liao et al. [1] on 420 patients showed that MRI was significantly superior to CT for detecting involvement of intracranial area, skull base, paranasal sinuses, oropharynx, parapharyngeal space, prevertebral muscle, and retropharyngeal node, resulting in changes of T-category in 50%, N-category 11%, and stage group in 39% of patients. In addition to more accurate delineation of gross tumor volume (GTV), this affected the decision on addition of chemotherapy in 20% of patients. Comprehensive search for distant metastases is indicated for patients with advanced locoregional disease, and those with suspicious clinical or laboratory abnormalities. Com­ parison of 4 modalities by Chua et al. [2] showed that integrated fluorodeoxyglucose positron emission tomography (FDG-PET) and CT was the most sensitive and specific modality for detecting distant metastases when compared with PET alone, CT thorax-abdomen plus skeletal scintigraphy, and conventional workup (chest X-ray, abdominal ultrasound plus skeletal scintigraphy): the corresponding sensi­tivity varied from 83 to 33%, and specificity from 97 to 90%. Ng et al. [3] showed that the total incidence of distant metastases

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Fig.  28.1  MRI showing different patterns of local and lymphatic involvement by nasopharyngeal carcinoma: (a) small primary tumor (white arrowhead); (b) extension into parapharyngeal space (star), ­prevertebral muscle (curved arrow), and clivus (black arrow); (c) the infiltrated foramen ovale (white arrow) as compared to the normal

opposite side (black arrow); (d) infiltration of cavernous sinus (white arrow) through foramen lacerum and direct skull base extension; (e) metastases in retropharyngeal nodes (white arrowheads); (f) metastases in Level II (black asterisks) and Level V cervical nodes (white asterisk)

detected by PET/CT was up to 14% among newly diagnosed patients, the treatment strategy was altered in 9% of patients (with correct modification of M-category and detection of second malignancy); PET/CT was also more accurate for detecting cervical nodes in 7% of patients; but it was inferior to MRI for delineating local infiltration and retropharyngeal nodes. Table 28.1 shows the staging criteria and groupings of the current staging system jointly used by American Joint Committee on Cancer [4] and International Union Against Cancer [5] (AJCC/UICC). It should be noted that the masticator space (one of the staging criteria for T4) is defined as a synonym of infratemporal fossa “extension of tumor beyond the anterior surface of the lateral pterygoid muscle, or lateral extension beyond the posterolateral wall of the maxillary antrum, and the pterygo-maxillary fissure” [4]. Unfortunately, different definitions are used in radiological textbook [6], inclusion of the medial and lateral pterygoid muscles as part of the masticator space will lead to unnecessary confusion and overtreatment. The TNM staging is the most important prognostic factor and the key factor in deciding treatment strategy, continual refinement to maximize predictive accuracy is needed. For further improvement of current AJCC/UICC system, the most

widely supported suggestion was down-staging of T2a to T1 and correspondingly subgroup T2N0 to Stage I [7–10]. One ambiguity in the current staging system is the categorization of retropharyngeal node(s). Detailed analyses of 924 MRI-staged patients by Tang et al. [11] showed that retropharyngeal node involvement was an independent factor for distant failure (p = 0.04), but there were no significant difference between unilateral and bilateral involvement (p = 0.73). The data thus suggested that retropharyngeal node involvement should be classified as N1, regardless of laterality. One additional investigation that helps to predict distant metastases and survival for non keratinizing cancer is the circulating Epstein–Barr Viral Deoxyribonucleic Acid (EBVDNA). Studies from endemic countries showed that high pretreatment copies of BamHI-W were associated with poor prognosis [12–15]. Leung et al. [12, 13] further showed that this could identify the high-risk subgroup among patients presenting with apparently early disease: 37% of Stage IIB with high copies developed distant metastases compared with none among those with low copies. This thus helps in identifying the poor risk subgroup for addition of chemotherapy. However, the methods for measuring circulating EBV-DNA have yet to be standardized and the cut off level for portending a worse prognosis have yet to be defined [16].

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Table 28.1  The Nasopharynx staging system by AJCC/UICC (7th edition) Primary tumor (T) TX Primary tumor cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ T1 Tumor confined to the nasopharynx, or tumor extends to oropharynx and/or nasal cavity without parapharyngeal extension a T2 Tumor with parapharyngeal extension a T3 Tumor involves bony structures of skull base and/or paranasal sinuses T4 Tumor with intracranial extension and/or involvement of cranial nerves, hypopharynx, orbit, or with extension to the infratemporal fossa/masticator space Regional lymph nodes (N): Nasopharynx The distribution and the prognostic impact of regional lymph node spread from nasopharynx cancer, particularly of the undifferentiated type, are different from those of other head and neck mucosal cancers and justify the use of a different N classification scheme NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Unilateral metastasis in cervical lymph node(s), 6 cm or less in greatest dimension, above the supraclavicular fossa, and/or unilateral or bilateral, retropharyngeal lymph nodes, 6 cm or less, in greatest dimension b N2 Bilateral metastasis in cervical lymph node(s), 6 cm or less in greatest dimension, above the supraclavicular fossa b N3 Metastasis in a lymph node(s) b >6 cm and/or to supraclavicular fossa b N3a Greater than 6 cm in dimension N3b Extension to the supraclavicular fossa c Distant metastasis (M) M0 No distant metastasis M1 Distant metastasis Anatomic stage/prognostic groups: Nasopharynx Stage 0 Tis N0 M0 Stage I T1 N0 M0 Stage II T1 N1 M0 T2 N0 M0 T2 N1 M0 Stage III T1 N2 M0 T2 N2 M0 T3 N0 M0 T3 N1 M0 T3 N2 M0 Stage IVA T4 N0 M0 T4 N1 M0 T4 N2 M0 Stage IVB Any T N3 M0 Stage IVC Any T Any N M1 Used with the permission of the American Joint Committee on Cancer (AJCC), Chicago, Illinois. The original source for this material is the AJCC Cancer Staging Manual, Seventh Edition (2010) published by Springer Science and Business Media LLC, http://www.springer.com a Parapharyngeal extension denotes posterolateral infiltration of tumor. b Midline nodes are considered ipsilateral nodes c Supraclavicular zone or fossa is relevant to the staging of nasopharyngeal carcinoma and is the triangular region originally described by Ho. It is defined by three points: (1) The superior margin of the sternal end of the clavicle. (2) The superior margin of the lateral end of the clavicle. (3) The point where the neck meets the shoulder. Note that this would include caudal portions of levels IV and VB. All cases with lymph nodes (whole or part) in the fossa are considered N3b

Primary Treatment

Radiation Therapy

Megavoltage radiation therapy (RT) has been the primary treatment modality. While excellent control can be achieved for patients with early disease, further improvement are needed for the majority of patients presenting with advanced locoregional diseases. The current recommendation is to treat patients with Stage I–IIA disease with RT alone, and those with Stage III–IVB (±IIB) disease with chemoradiotherapy (CRT).

Dose, Time, and Fractionation Although NPC is a radiosensitive tumor, high dose is needed for complete eradication. Retrospective studies showed a significant dose–response effect [17–19]; total dose ³70 Gy is needed even for T1–2 tumors. Fractional dose did not affect local control, but it was a significant risk factor for temporal

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lobe necrosis [20–22]. The general recommendation is to give 2 Gy per daily fraction to a total dose ³70 Gy to gross tumor, and 50–60 Gy for elective treatment of potential risk sites. Leung et  al. [23] showed that for patients with T1–2b tumors, an additional boost by high-dose-rate (HDR) brachytherapy of 10–12 Gy in 2 weekly fractions following 66 Gy by 2-dimensional (2D) RT could achieve significantly better 5-year L-FFR (96% vs. 88%) and overall survival (OS) (91% vs. 80%) without excessive late toxicity (10% vs. 14%) when compared with historic controls. Study by Hara et al. [24] on 82 patients (52% with T3–4 tumor) showed that a single-fraction of 7–15  Gy by stereotactic RT following an external course of 66 Gy together with extensive use of concurrent-adjuvant chemotherapy in advanced cases, could achieve excellent 5-year L-FFR of 98%. However, relatively high incidences of late damage (including temporal lobe 12%, retina 4%, and carotid artery 1%) had been observed. The therapeutic risk and benefit of dose escalation, particularly for patients treated with CRT, have to be cautiously balanced. Retrospective study showed that prolongation of treatment significantly jeopardized local control [25, 26], the hazard of local failure increasing by 3% per additional day even for non keratinizing tumor. However, the benefit of accelerated fractionation (AF) was uncertain. Teo et al. [27] randomized 159 patients (38% T3–4) to 2.5 Gy/fraction daily (QD) for 8 fractions followed by 1.6 Gy twice-daily (BID) for another 32 fractions vs. 2.5 Gy/fraction daily (QD) for 24 fractions, the 5-year L-FFR was 89% vs. 85%, but excessive neurological toxicities were incurred (49% vs. 23%). Daoud et al. [28] randomized 154 patients (45% T3–4) to 1.6 Gy/ fraction BID to 70.4  Gy/6 weeks vs. 2  Gy/fraction QD to 70  Gy/7 weeks; the 5-year locoregional control (LR-FFR) was 81% vs. 78%; no major excessive toxicities were observed. The preliminary results of NPC-9902 Trial by Lee et al. [29] on 198 patients with T3-4N0-1M0 diseases comparing 5 vs. 6 fractions per week at 2 Gy per fraction also showed that AF per se was disappointing, but AF combined with concurrent-adjuvant chemotherapy achieved significantly

better event-free survival (EFS) than RT at conventional fractionation (CF) alone (94% vs. 70% at 3-year; p = 0.008); further confirmation is warranted.

Tumor Targets and Technique The delineation of gross tumor volume (GTV) should be based on all clinical, endoscopic, and imaging findings. Fusion of diagnostic MRI and PET (if available) with planning CT is valuable for accurate delineation of tumor targets, but setting of most accurate segmentation for PET can be difficult [30, 31], close collaboration with diagnostic radiologist and nuclear physician is important. The clinical target volume (CTV) covers the GTV and microscopic infiltration, including anatomical structures at risk. There is little controversy that intensity-modulated technique (IMRT) is recommended if resources permit, dosimetric studies showed clear improvement in conformity of dose distribution for the complex concave tumor targets and better protection of the adjacent organs [32–35]. Different centers have different practices regarding the delineation of CTV, prescription of dose fractionation, priorities in dose constraints, acceptance criteria, and beam arrangements. Many use simultaneous boost to deliver AF: the dose/fraction to the GTV ranged from 2.12 Gy [36, 37] to 2.34 Gy [38]. Together with additional chemotherapy and/or enhanced RT with boosts or AF, all series using IMRT reported most encouraging early results with local control in excess of 90% at 2–4 years (Table 28.2). Randomized trials on patients with early stages showed significant sparing of parotid glands [39, 40]. However, it should be cautioned that overenthusiasm in protecting the parotids might result in marginal miss. Furthermore, late toxicities at other sites still developed (see Section on Late Toxicities), stringent dose constraint to organs at risk (OAR) is needed. Caution on the risk of high doses to the larynx [41], mandible

Table 28.2  Treatment parameters and outcome by intensity-modulated radiotherapy Author (reference) Lee [36] Kwong [155] Kam [142] Wolden [38] Kwong [140] No. of patients T3–4 category (%) Total dose (Gy) Dose/fraction (Gy) Acceleration (%) Additional boost (%) Chemotherapy (%) Tumor control Time (years) Local control (%) Nodal control (%) Distant control (%) Overall survival (%)

Lin [156]

Tham [157]

Lee [37]

PYNEH

67 43 65–70 2.12–2.25 0 39 75

50 0 68–70 2–2.06 0 0 0

63 51 66 2 0 56 30

74 51 70.2 2.34 80 0 93

50 100 76 2.17 0 0 68

326 61 66–69.75 2.2–2.25 0 20 90

195 NS 70 2.12 0 10 57

68 34 70 2.12 0 0 84

193 93 70 2 62 0 84

4 97 98 66 88

2 100 94 94 –

3 92 98 79 90

3 91 93 78 83

2 96 98 94 92

3 95 98 90 90

3 90 – 89 94

2 93 91 85 80

2 95 96 90 93

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[42], pharyngo-esophageal axis [43], brachial plexus [44], and the carotid arteries [45] should be noted. Depending on the disease extent, sparing of other normal structures such as the submandibular gland, soft palate, middle, and inferior constrictor muscles should also be attempted if feasible in order to reduce xerostomia, acute odynophagia/velopalatine insufficiency, and dysphagia, respectively,

Treatment Precision Image guidance to ensure precision in treatment delivery is a major advance. Electronic treatment position verification devices allow daily imaging and online correction of positional errors prior to treatment. Orthogonal kV images to verify position of bony landmarks and recent development of online cone-beam CT-guided IMRT [46] further improve the accuracy of treatment delivery. Interfractional anatomic changes due to tumor shrinkage and weight loss could adversely affect the ultimate doses delivered [47, 48], these changes could significantly increase the daily maximum dose to the neighboring critical structures including brain stem, spinal cord, and optic chiasm. Although the maximum dose point might not fall into the same part of the critical structures to cause damage, close

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monitoring is needed; re-planning should be considered if marked deviation in dose distribution occurs.

Example of Planning and Treatment Practice The current practice at the Pamela Youde Nethersole Eastern Hospital (Hong Kong) is described as an example. The patient is set up in a supine position with head extended, and immobilized by a customized thermoplastic mask covering the head to shoulder regions. A contrast CT is taken in treatment position covering from skull vertex to 2 cm below clavicles, with 3  mm slice thickness at gross tumor regions is performed. The diagnostic MRI and PET (if available) are co-registered with planning CT for delineation of tumor targets and OAR. The whole-volume is irradiated by 9–11 (mostly coplanar) 6-MV photon beams using IMRT technique with dynamic multileaf collimator (MLC). Figure 28.2 illustrates the targets for patients with different locoregional involvement. A total dose of 70 Gy at 2 Gy/ fraction is prescribed to CTV1 that aims to include the primary tumor with a 2–5 mm margin, the whole nasopharynx, and gross lymph nodes with a 5–10 mm margin. Instead of using simultaneous boost, patients with T3–4 tumors are treated with a moderate AF schedule of 6 daily fractions per

Fig. 28.2  Delineation of tumor targets for different locoregional involvement as listed above: the purple line for the gross tumor volume (GTV), the red line for the clinical target volume (CTV) aimed at 70 Gy, and the orange line for the CTV aimed at 61.25 Gy

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week. The CTV2 that aims to cover high-risk structures (including parapharyngeal spaces, posterior third of nasal cavities and maxillary sinuses, pterygoid processes, base of skull, lower half of sphenoid sinus, anterior half of the ­clivus, petrous tips, bilateral retropharyngeal nodes, Levels II, III, and upper VA lymphatic regions) receive 61.25 Gy at 1.75 Gy/fraction. By reducing the field borders for the last 5 fractions, the CTV3 that covers low-risk structures including the remaining lymphatic Levels IV–VB and upper half of sphenoid sinus (if T3–4 tumor) receive 52.5 Gy also at 1.75  Gy/fraction. Selective sparing of level IB is considered in N0 patients. The CTV are expanded by 3  mm to constitute the planning target volume (PTV) for systemic and random set up variation. With such a tight margin, image guidance to ensure setup accuracy (Fig. 28.3) is indicated particularly for patients with T4 tumors. Daily orthogonal kV images are taken to verify position of bony landmarks and correction applied for deviations ³2 mm. Weekly fusions of cone-beam CT with deformable registration is currently under studied to assess the dosimetric variation due to anatomical changes during the RT course. Treatment is re-planned if the dose distribution deviates significantly from the intended plan. Table 28.3 shows the acceptance criteria for Inverse Planning. Top priority is given to critical neurological structures, followed by tumor targets, organs with intermediate importance, and finally those with lower importance. Optimization to achieve the ideal criteria is attempted as far as possible; best possible balance between tumor control and toxicity is discussed with individual patient if it is technically difficult to fulfill even the minimal requirement. From 2005 to 2007, 193 patients (93% with stage III–IVB disease) had been treated with the above described technique in our center. Accelerated fractionation was used in 62%, and additional chemotherapy was given to 84% of patients. The median follow-up was 30 months (range, 4–45 months). The 2-year L-FFR, nodal failure-free rate (R-FFR), D-FFR, and OS were 95, 96, 90, and 93%, respectively. Review of doses given to the locoregional failure sites showed that 4 failures were marginal miss: failure occurred at bilateral cavernous sinus (1%), maxillary and ethmoid sinuses (0.5%), and submental node (0.5%). Longer follow-up is needed to assess the ultimate efficacy and late toxicities.

Technological Developments Development of helical tomography (HT) capable of calculating MLC position every 7 degrees of rotation opened a new opportunity for achieving more uniform target dose. Dosimetric comparisons showed that HT was superior to coplanar 5-fields IMRT delivered by dynamic MLC [49] and 7-fields step-and-shoot IMRT [50] in the homogeneity of

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Fig.  28.3  Verification of setup accuracy: (upper) simulator checkfilms; (mid) checking of bony landmarks using On Broad Imager™; and (lower) cone-beam computed tomography to ensure no major deviation

dose distribution within the PTV; significant improvement ratios of 129% in conformity index and 9% in homogeneity index was shown by Lee et al. [50]. In addition, significant reduction of mean/maximal doses to most of the OAR was achieved, but it should be noted that negative result was observed for optic chiasm, particularly for patients with T1–2 tumor, due to less sharp fall-off in craniocaudal dose. It is hoped that recent development of volumetric arc technique using linear accelerator can achieve improvement in dose distribution with greater efficiency in delivery. Particle beam RT (hadrontherapy) with physical advantages of better spatial selectivity and/or higher biological

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Table  28.3  Intensity-modulated radiation therapy for nasopharyngeal carcinoma: specification on dose constraints and acceptance criteria (current guideline at Pamela Youde Nethersole Eastern Hospital, Hong Kong) First criteria – ideal Second criteria – acceptable Priority 1: critical organ at risk Brainstem Spinal cord Optic chiasma Optic nerve Temporal lobes

Point <54 Gy Point <45 Gy Point <54 Gy Point <54 Gy Point <65 Gy & 1% volume <60 Gy

Point & 1% volume <60 Gy Point & 1cc volume <50 Gy Point & 1% volume <60 Gy Point & 1% volume <60 Gy Point <70 Gy & 1% volume <65 Gy

Priority 2: tumor targets GTV PTV

³68.6 Gy (98% dose) to 100% PTV1 ³95% dose to 100% PTV <10% PTV1 ³75 Gy (107% dose)

<1% GTV <66.5 Gy (95% dose) <5% PTV <100% dose <1% PTV <93% dose <20% PTV1 ³77 Gy (110% dose)

Point <60 Gy 1% volume <70 Gy Point <6 Gy Point <50 Gy

1% volume <65 Gy 1% volume <75 Gy Point & 1% volume <10 Gy Mean <35 Gy

Priority 3: organ at risk with intermediate importance Pituitary Mandible/T-M joint Lens Eyeball

Priority 4: organ at risk with low importance Parotid glands Mean <26 Gy (at least 1 gland) Cochlea Mean <50 Gy Tongue 1% volume <70 Gy Larynx Mean <30 Gy Nonspecified <1% volume ³75 Gy GTV gross tumor volume, PTV planning target volume, PTV1 aimed at 70 Gy

efficacy than photons is an attractive technological advance for NPC with its critical location. Dosimetric comparisons showed that 3D spot-scanned coplanar 3-fields intensitymodulated proton therapy (IMPT) was superior to coplanar 9-fields step-and-shoot IMRT by photon [51] in coverage and conformity for the GTV, as well as reduction of mean dose to most OAR and medium dose volumes by a factor of 2–3. The latter improvement might be important for minimizing late damages and carcinogenic effect of ionizing radiation. Further comparative study by Widesott et al. [52] showed that both IMPT and HT could achieve similar coverage and homogeneity for PTV; the most remarkable superiority of IMPT was reduction in total body volume receiving ³30–10 Gy by 15–23%. Selective use of this expensive treatment for extensive locoregional disease infiltrating/abutting critical OAR and re-irradiation of recurrent tumor, particularly for pediatric patients, is worth exploring.

Chemotherapy Current Recommendations The meta-analysis by Baujat et al. [53], based on 1,753 patients from trials on induction chemotherapy [54–57], adjuvant chemotherapy [58], and concurrent ± adjuvant chemotherapy [59–61], confirmed that significant survival benefit could be achieved by adding chemotherapy: The absolute gain for 5-year

50% volume <30 Gy (at least 1 gland) – Mean dose <55 Gy Mean <45 Gy <5% volume ³70 Gy

EFS was 10% (52% vs. 42%) and OS was 6% (62% vs. 56%). Concurrent chemotherapy was the most potent combination, and the only sequence that achieved significant benefit in OS: hazard ratio (HR) = 0.71; 95% confidence interval (CI), 0.53–0.94. Induction chemotherapy per se could significantly reduce the risk of locoregional failures by 24% and distant failures by 35%; this resulted in significant improvement in EFS (HR = 0.82; 95% CI, 0.68–0.97), but the benefit in OS was nonsignificant (HR = 0.99; 95% CI, 0.80–1.21). Adjuvant chemotherapy per se failed to achieve significant benefit in any endpoints. Table 28.4 summarizes the trials on concurrent ± adjuvant chemotherapy published in the English literature. The first trial that achieved significant survival benefit was the Intergroup-0099 Study [59] using cisplatin (CDDP) (100 mg/m2) on days 1, 22, and 43 in concurrence with RT at conventional fractionation (70  Gy in 35 fractions) followed by combination of CDDP (80 mg/m2) and fluorouracil (FU) (1,000 mg/m2/day for 96 h) on days 71, 99, and 127 during the post-RT phase. Subsequent confirmatory trials by Lee et al. [62], Wee et al. [63], and Chen et  al. [64], consistently supported that the Intergroup-0099 regimen could significantly improve tumor control; the latter two trials also showed significant survival benefit. In addition, the NPC-9902 Trial (on patients with T3–4N0–1 disease) by Lee et  al. [29], showed that the concurrent-adjuvant chemotherapy was a significant independent factor for reducing relapse (HR = 0.52; 95% CI, 0.28–0.97; p = 0.039); furthermore, the preliminary results suggested that combining this regimen with accelerated fractionation could achieve substantially greater benefit.

3 –

P PF

P PF

P PF

mean 69 C P PF

70 C U ± PF/VBM

PF –

70–74 C

284 II–IVB

Lin [65]

P –

66 C

350 II–IVB

Chan [61]

O –

70–74 C

115 III–IVB

Zhang [67]

3 3 2 3 5 5 2 92 vs. 82 81 vs. 85 94 vs. 85 98 vs. 92 80 vs. 72 L: 89 vs. 73 NS L: 96 vs. 88 p = 0.01 NS NS p = 0.01 NS p < 0.01 NS Distant control S 87 vs. 70 a 76 vs. 73 89 vs. 81 97 vs. 81 87 vs. 79 85 vs. 71 79 vs. 70 NS 92 vs. 80 p < 0.01 NS NS p = 0.03 p = 0.02 p = 0.03 (p = 0.06) p = 0.02 Progression-free survival 58 vs. 29 72 vs. 53 70 vs. 61 73 vs. 68 88 vs. 68 – – – – 96 vs. 83 p < 0.01 p = 0.01 NS NS (p = 0.06) (p = 0.09) Failure-free survival – – 72 vs. 62 74 vs. 70 94 vs. 70 85 vs. 73 69 vs. 58 72 vs. 53 60 vs. 52 – p = 0.03 NS p = 0.01 p < 0.01 NS p = 0.01 NS Overall survival 67 vs. 37 80 vs. 65 78 vs. 78 87 vs. 83 88 vs. 83 90 vs. 80 87 vs. 77 72 vs. 54 70 vs. 59 100 vs. 77 p < 0.01 p = 0.01 NS NS NS p < 0.01 (p = 0.06) p < 0.01 (p = 0.07) p = 0.01 a  2-year incidence of freedom from distant failure as the first site of failure AJCC/UICC 6th , American Joint Committee on Cancer/International Union Against Cancer 6th Edition; C, conventional; A, accelerated; P, cisplatin; F, fluorouracil; U, uracil-tegafur; V, vincristine; B, bleomycin; M, methotrexate; O, oxaliplatin; L, local; S, significant but no detailed data; NS, no statistical significance (p > 0.1); Progression-free survival: defining events included both failures and death; Failure-free survival: defining events included failures only

Tumor control (%): CRT vs. RT Time point (year) 5 Locoregional control S

P PF

mean 68 C

62.5–68 C

Chemotherapy Concurrent Adjuvant

70 C A

70 C

316 III–IVB

Radiotherapy Total dose (Gy) Fractionation

189 III–IVA

222 II–IVB

348 III–IVB

193 II–IVB

Patient characteristics Number enrolled Stage (AJCC/UICC 6th) 221 III–IVB

Kwong [60]

Table 28.4  Randomized trials comparing concurrent chemoradiotherapy vs. radiotherapy alone for nasopharyngeal carcinoma Author (reference) Al-Sarraf [59, 74] Wee [63] Lee [62] Lee [29] Chen [64]

388 A.W.M. Lee et al.

28  Management of Nasopharyngeal Carcinoma

389

The trials using concurrent chemotherapy alone showed less consistent conclusions. Lin et  al. [65] using concurrent CDDP/FU reported significant benefit in both EFS and OS. However, subsequent re-analysis [66] with retrospective restaging of the accrued patients into different risk groups showed that the benefit was significant for low-risk patients only. The trial by Chan et al. [61] using concurrent weekly CDDP and that by Kwong et al. [60] using concurrent uracil-tegafur with or without adjuvant CDDP-based combination only showed borderline improvement in OS (p > 0.06) and no significant improvement in failure rate (p > 0.14). A trial by Zhang et al. [67] using concurrent oxaliplatin showed significant improvement in both EFS and OS at 2-year, longer results are awaited. A randomized trial by Chitapanarux et  al. [68] on 206 patients showed that replacing CDDP in the Intergroup-0099 regimen with carboplatin could lead to better tolerability (proportion of patients who could complete 6 cycles was 62% vs. 26%) with similar efficacy (3-year OS was 78% vs. 79%). However, it should be noted that the completion rate in the CDDP-group was very low. Our retrospective study comparing patients who completed two cycles of concurrent chemotherapy [69] showed that those with both cycles replaced by carboplatin had significantly inferior 3-year LR-FFR (56% vs. 86%, p = 0.014) and OS (61% vs. 87%, p = 0.046). Retrospective comparison of concurrent-adjuvant chemotherapy vs. concurrent chemotherapy alone in stage I–III patients by Cheng et al. [70] showed that the former could achieve significantly better EFS (77% vs. 53% at 5-year, p =0.01), the adjuvant phase was beneficial particularly for patients with T2b–3 N0–2 diseases. Hence, basing on currently available data, the Intergroup-0099 regimen remains

the standard chemotherapy recommended for patients with advanced locoregional disease outside clinical trials. However, there are concerns about the efficacy of the Intergroup-0099 regimen for distant control. Preliminary results of the NPC-9901 Trial [62] showed little improvement in distant control for patients with N2–3 disease (76% vs. 73% at 3-year; p = 0.47). Reports from Stanford University [24] and University of California, San Francisco also showed that the incidence of distant failure remained high (>30%) despite achievement of excellent locoregional control by new technologies and extensive use of the Intergroup-0099 regimen (>75% of the series). Exploration for a more potent strategy is needed.

New Strategies Under Investigation One logical strategy is to change the sequence of the Intergroup-0099 regimen from concurrent-adjuvant to induction-concurrent because the induction sequence is more potent for reducing failures and substantially better tolerated. Theoretically, early use of potent combination of cytotoxic drugs at full dose would be more effective for eradicating micrometastases. Another possible advantage is that this could shrink the primary tumor to give wider margin for irradiation, an advantage that is particularly needed for patients with extensive locoregional infiltration infiltrating/abutting critical neurological structures. Table  28.5 summarizes the reported Phase II studies on induction-concurrent CRT. The first study on 35 patients

Table 28.5  Phase II studies on induction-concurrent chemoradiotherapy Author (reference) Rischin [71] Oh [158] Johnson [161] Al-Amro [160]

Chan [79]

Hui [76]

Patient characteristics Number studied Stage IV (%)

35 40

27 NR

44 NR

110 74

31 39

34 44

Radiotherapy Total dose (Gy) Overall time (weeks)

60 6

70 14 a

70 7

66 6.5

66 6.5

66 6.5

Chemotherapy Induction Concurrent

PEF P

PFI HF

PF PF

PE P

TJ P

DP P

Tumor control (%) Time point (year) Locoregional control Distant control Event-free survival

4 L: 97 94 81

5 93 92 86

5 75 b 89 b 55

3 68 74 53

2 90b 81b 79

Overall survival

90

77

66

71

92

3 – – 88 vs. 64 NS 94 vs. 68 p = 0.01

31 39

– P

Lee [72]

Yau [73]

49 100

37 100

70 6

70 6

PF P

PG P

3 77 75 61

3 78 76 63

71

76

Split fractionation (2 Gy/fraction daily × 5 fraction, q 2 week) Crude incidence L, local failure-free rate alone; P, cisplatin; D, docetaxel; F, fluorouracil; E, epirubicin; I, interferon-a; H, hydroxyurea, T, paclitaxel; J, carboplatin; G, gemcitabine

a

b

390

(40% with stage IV disease) by Rischin et al. [71] achieved excellent 4-year LR-FFR 97%, D-FFR 94%, and OS of 90%. All studies except that by Rischin et al. [71] aimed at a total radiation dose of 66–70  Gy. Additional enhancement by accelerated fractionation was attempted in our two studies that focused on patients with stage IVA–B disease [72, 73]. Different chemotherapy regimens have been tested; crossseries comparison is not possible because of marked variation in patient characteristics. Experience from our center [72] on changing the sequence of the Intergroup-0099 regimen to three cycles of CDDP/FU as induction chemotherapy followed by two cycles of CDDP in concurrence with accelerated RT showed that patients did indeed had substantially better tolerance and compliance to induction chemotherapy: 98% of patients could complete three cycles of induction chemotherapy, even with a scheduled 20% increase in the dose intensity of CDDP and FU, whereas only 55% of patients in the Intergroup-0099 Trial [59] completed three cycles of adjuvant chemotherapy. Only 2% of patients had early termination of induction chemotherapy because of poor response. The induction treatment did not substantially jeopardize the tolerance during the concurrent phase, 96% of patients could complete the whole course of RT with a median overall treatment time of 41 days, only 7% failed to complete two cycles of concurrent chemotherapy. Using this chemotherapy sequence and accelerated 3D conformal RT for patients with stage IVA–B disease, our first study showed encouraging results of LR-FFR 77% and OS 71% at 3 years. In a subsequent study [75] using IMRT technique, we further showed that induction chemotherapy using the CDDP/ FU regimen could achieve significant down-staging of T-category (p = 0.016): 25% of T3–4 tumors became T1–2 and another 10% decreased from T4 to T3. Furthermore, this could significantly reduce the GTV_P (primary tumor volume) by an average of 61%, leading to increase in minimum dose from 62.3 to 64.5 Gy (p < 0.020), decrease in volume within GTV_P that failed to reach 70 Gy from 10.2 to 3.8%, and consequent improvement in the estimated tumor control probability (mean value) from 0.83 to 0.89 (p = 0.002). With a median follow-up of 14 months, only 1/20 patient died of distant failure, while the remaining 95% were alive without locoregional failure; longer follow-up is needed to confirm the long-term treatment efficacy. Although this strategy using CDDP/FU is effective and the treatment toxicity is acceptable, continuous infusion of FU for 120 h is very inconvenient, requiring frequent hospitalization or central access with insertion of infusion pump for administration. An effective regimen that is more convenient and less toxic will be desirable. Hence, we have also tried to replace FU with gemcitabine as induction chemotherapy [69, 73], retrospective comparison of the two induction regimens (CDDP/gemcitabine vs. CDDP/FU) in 75

A.W.M. Lee et al.

patients with stage IVA–B disease treated with the same accelerated CRT schedule showed no significant differences in all tumor control endpoints: the 3-year EFS were almost identical (61% vs. 66%, p = 0.997); although the LR-FFR was slightly higher in the CDDP/gemcitabine Group (85% vs. 76%, p = 0.310), no improvements in D-FFR (78% vs. 83%, p = 0.310) and OS (70% vs. 85%, p = 0.310) were achieved. It seemed that this regimen is likely to be equally effective, but not superior to the CDDP/FU regimen. A randomized Phase II trial [76] on 65 patients with stage III–IVB disease showed that those treated by induction chemotherapy using CDDP/docetaxel followed by concurrent CRT using weekly CDDP achieved significantly higher OS (94% vs. 68% at 2-year; p = 0.012) than those treated by concurrent CRT alone, though the improvement in EFS did not reach statistical significance (88% vs. 60%; p = 0.12). It should be cautioned that this induction regimen incurred Grade 3–4 neutropenia in 97% of patients, with 12% neutropenic fever. Induction-concurrent CRT is hence a promising strategy for improving tumor control and there are two on-going randomized trials to evaluate this strategy. The GORTECNPC2006 Trial by Groupe Oncologie Radiothérapie Tête et Cou focuses on patients with stage II–IVB disease: the standard arm is weekly CDDP in concurrence with RT at conventional fractionation; the aim is to evaluate the benefits achieved by adding Docetaxel/CDDP/FU as induction chemotherapy. The NPC-0501 Trial by the Hong Kong Nasopharyngeal Cancer Study Group focuses on patients with stage III–IVB disease: the standard arm is the Intergroup-0099 regimen using concurrent CDDP plus adjuvant CDDP/FU with RT at  conventional fractionation. The aims are to compare the therapeutic benefits achieved by changing the chemotherapy sequence from concurrent-adjuvant to induction-concurrent and changing the RT schedule from conventional to accelerated fractionation. In addition, this trial attempts to study the  possibility of replacing FU with the oral pro-drug capecitabine.

Monitoring of Progress Early detection of treatment failure is crucial for better chance of salvage; both endoscopic and imaging examinations are needed. A systemic review of 1,813 patients from published literature [77] showed that FDG-PET is the best modality for diagnosis of persistent/recurrent locoregional disease. Both the sensitivity and specificity estimates for PET (95 and 90%) were significantly superior to MRI and CT (p < 0.001); the sensitivity of MRI and CT were similar (78 and 76%, respectively), but the specificity for MRI was significantly better than CT (76% vs. 59%, p < 0.001).

28  Management of Nasopharyngeal Carcinoma

Another useful investigation for monitoring disease is the circulating plasma EBV-DNA, patients with persistently elevated posttreatment levels had a significantly higher risk of relapse and death than those without [15, 16, 78–80]. Chan et  al. [78] showed that the relative risk for recurrence was 11.9-fold for patients with persistently raised plasma EBV-DNA at 6–8 weeks posttreatment. Longitudinal follow-up showed that in 89% (8/9) of patients who developed treatment failure, the EBV-DNA level started to increase 2–16 months before clinical evidence of disease progression [79].

Treatment of Persistent/Recurrent Tumors As it takes time for tumors to regress following RT, one difficult decision is when to consider residual tumors as genuine persistence and proceed with salvage treatment. Kwong et  al. [81] showed that the incidence of positive histology decreased spontaneously from 29% in the first week after completion of RT to 12% by the ninth week and then rose again. The 5-year L-FFR was 82% for patients who achieved early histological remission (<5 weeks), 77% for those with delayed remission (5 to <12 weeks), but only 54% for those with persistent tumors at 12 weeks despite subsequent salvage treatment. The optimal time for intervention remains uncertain; avoidance of unnecessary overtreatment and excessive delay in treatment are both important considerations, treatment decision basing on findings between 8 and 12 weeks is a reasonable balance [82–84]; but some studies were based on biopsies taken between 3 to 8 weeks [85, 86], the time of intervention of “persistent disease” has to be taken into account in interpreting the treatment results. Because the therapeutic considerations and prognosis are different, distinction should be made between persistent disease (tumors that do not completely regress following primary treatment) and recurrent disease (tumors that re-emerge after initial complete regression). Patients with persistent disease had better outcome than those with recurrent disease. Wu et  al. [87] showed that the timing and the volume of tumor detected were independent prognostic factors, the 3-year disease-­specific survival (DSS) was significantly higher for the per­sistent group (patients who failed within 6 months from completion of RT) than the recurrent group: 81% vs. 46% (p = 0.037).

Nonsurgical Salvage Table  28.6 summarizes the recent reports on different RT methods and the outcome for the two groups. Brachytherapy,

391

using intracavitary and interstitial methods, has been widely used for superficial persistent diseases. Excellent results with 5-year L-FFR of 90% and above have been reported for patients with initial T1–2 tumors [82, 83, 85, 86]. To ensure adequate coverage of more bulky tumor and precise delivery, stereotactic RT is increasingly used. Various dose prescriptions have been used, ranging from 7–35  Gy by single fraction radiosurgery (SRS) [88, 89] to  10–24  Gy by multiple fractions have been employed [84, 87]. Yau et al. [84] showed that 7% of 755 consecutive patients had positive biopsies 8 weeks after completion of primary RT: those treated with fractionated stereotactic RT (FSRT) to a median dose of 15  Gy achieved a 3-year L-FFR of 82%, a result that was very close to corresponding 86% in the contemporary cohort with complete remission, and was substantially better than corresponding L-FFR of 71% in those treated with HDR-brachytherapy to a median dose of 20  Gy. Wu et  al. [87] also reported 3-year L-FFR of 89% with FSRT, and they further showed that severe late toxicity rate (9%) was substantially lower than SRS series. For patients with local recurrence, the TNM stage of the recurrent tumor is one of the most important prognostic factors. Thorough re-staging workup is needed as 54% of patients also had synchronous regional and/or distant failures [90]. For the majority with recurrence infiltrating beyond the nasopharynx, re-irradiation with or without chemotherapy is the only option. Aggressive treatment should be attempted as far as possible because long-term survival might be achieved [91]; but doses ³60  Gy are needed for effective salvage [92–94], significant morbidities are often incurred. Re-irradiation poses a therapeutic challenge, it is crucial to restrict the irradiation of normal tissue to a minimum. A retrospective study by Lee et al. [95] comparing the late toxicity rate in 487 patients with two courses of external RT vs. 3,635 patients with one course suggested that there was partial recovery of normal tissues following the primary course: the summated total biological dose tolerated (BEDS(sigma)) was higher than that expected with a single course treatment (BED-1). Assuming an a(alpha)/b(beta) ratio of 3 Gy, the BED-S(sigma) that incurred 20% toxicity at 5-years was 129% that of BED-1. Brachytherapy has been widely used for treatment of superficial recurrent disease. Using interstitial implants with radioactive gold grains, Kwong et al. [82] reported a 5-year L-FFR of 63%; complications included headache (28%), palatal fistula (19%), and mucosal necrosis (16%). Using iridium mold, Law et al. [83] achieved excellent local ­salvage of 89%, but the complication rate was 53%. For more advanced recurrent tumors, addition of brachytherapy boost to external RT with 2D technique could achieve higher salvage rate [92–94, 96].

392

A.W.M. Lee et al.

Table 28.6  Radiotherapy for salvage of persistent/recurrent nasopharyngeal carcinoma Major late toxicity (Cumulative %) Overall TLN

79 65 NR

28 14 NR

NR NR 0

NR

21

2

82 NR

27 9

NR 0

54 65 NR rT1: 39 rT2: 24 rT3: 28 rT4: 4 26 rT1: 70 rT2: 52 rT3: 32 rT4: 10 63

19 53 NR 2D: 23 3D: 9

NR NR 33 2D: 14 3D: 0

Author

Treatment

Treatment outcome (Actuarial rate %) Year L-FFR Survival

Persistence Kwong et al. [82] Law et al. [83] Leung et al. [85]

Interstitial gold grain Iridium mold HDR-ICB

5 5 5

Zheng et al. [86]

HDR-ICB

5

Yau et al. [84] Wu et al. [87]

FSRT FSRT

3 3

T1: 87 T1–2a: 90 T1: 95 T2: 88 T1: 100 T2: 90 T1–4: 82 T1–4: 89

Recurrence Kwong et al. [82] Law et al. [83] Leung et al. [96] Chang et al. [102]

Interstitial gold grain Iridium mould 2D-RT + HDR-ICB 81% 2D, 19% 3D

5 5 3 3

rT1: 63 rT1–2a: 89 rT1–2: 72 NR

Poon et al [108] Zheng et al. [104]

Concurrent-adjuvant chemo + RT All 3D

5 5

15 24 3 rT1: 92 49 16 rT2: 81 rT3: 68 rT4: 41 Chua et al. [106] IMRT 1 rT1–3: 100 19 7 rT4: 35 Chua et al. [107] Induction chemo + IMRT/SRS 1 rT2–3: 100 88 24 12 rT4: 52 Chua [101] SRS 3 rT1–3: 68 78 22 14 Leung et al [100] FSRT 5 57 40 57 20 L-FFR local failure-free rate, TLN temporal lobe necrosis, HDR-ICB high-dose-rate intracavitary brachytherapy, RT radiotherapy, FSRT fractionated stereotactic radiotherapy, SRS stereotactic radiosurgery, 2D 2-dimesional, 3D 3-dimesional conformal, IMRT intensity-modulated radiotherapy

Stereotactic RT is increasingly used; local salvage rates ranging from 53 to 86% have been reported [88, 97–100]. Leung et  al. [100] showed that the total equivalent dose (TED) by FSRT was a significant factor, TED ³55 Gy was recommended. For patients with limited local failure, both SRS and gold grain implantation yielded comparable high tumor control rates [101]. A higher salvage rate by adding stereotactic RT as a boost after external RT has been reported [98, 102, 103]. Although, most series reported a low risk of complications, torrential hemorrhage with potential fatal outcome has been reported [98]; radiosurgery should be avoided in patients with tumor encasing the carotid artery or previous high cumulative dose. Past series using 2D technique achieved 5-year survival rates in the range of 21–41%, while the incidence of temporal lobe necrosis (TLN) ranged from 2 to 27%. The use of 3D conformal radiotherapy showed improving results. Chang et al. [102] showed that none of the patients re-irradiated by

3D technique developed TLN compared to 14% in those by 2D technique. Zheng et al. [104] reported a very encouraging 5-year local salvage rate of 71%, but grade 4 late toxicity rate was still as high as 49%. Preliminary reports using IMRT for re-irradiation show encouraging preliminary results. Using IMRT to deliver 68–70 Gy, Lu et al. [105] reported 100% salvage rate without any severe late complications in a series of 49 patients with a median follow-up of 9 months. Using IMRT to a median dose of 54 Gy in 31 patients (with or without induction chemotherapy and stereotactic boost), Chua et al. [106] reported a locoregional salvage rate of 56% and late grade 3 complications of 25% at 1-year. Longer follow-up is clearly needed. Chemoradiotherapy may also improve treatment ­outcome for recurrent NPC. Using CDDP/gemcitabine as induction chemotherapy followed by re-irradiation with IMRT in 20 patients (95% rT3–4), Chua et al. [107] reported a 1-year

28  Management of Nasopharyngeal Carcinoma

local salvage rate of 75%. Using concurrent CDDP followed by adjuvant chemotherapy with CDDP/FU in 35 patients (66% rT3–4), Poon et al. [108] reported a 1-year EFS of 42%.

Surgical Salvage For patients who develop locoregional failure despite aggressive primary treatment, surgical salvage is one of the options to be considered. In patients with nodal failure, the extent of involvement is often extensive; radical neck dissection should be carried out as the salvage procedure, 5-year nodal salvage of 66% and EFS of 37% could be achieved. For those with extranodal infiltration, addition of after-loading brachytherapy to the tumor bed following radical neck dissection could achieve similar results [109–111]. For patients with local failure, nasopharyngectomy is an option for selected patients with localized disease. Different approaches have been employed to expose the nasopharynx for oncological extirpation of the tumor [112–115]. At the University of Hong Kong Medical Centre, we employ the anterolateral approach or the maxillary swing procedure: ­following facial incisions and osteotomies, the maxilla bone is swung laterally while attached to the anterior cheek flap as one osteocutaneous unit (Fig.  28.4). The nasopharynx and its vicinity including the parapharyngeal space could hence be widely exposed to enable adequate resection of the persistent/recurrent tumor; the maxilla is returned and fixed to the rest of the facial skeleton with mini-plates at completion of the nasopharyngectomy. From 1989 to 2008, we have performed this salvage nasopharyngectomy with curative intent for 161 patients

393

with recurrent T1 disease. During resection, negative tumor resection margins with frozen section were achieved in 78% of patients, while the remaining had microscopic residual tumor beyond the scope of further resections. All patients recovered from the operation and were discharged. Associated morbidities included a varying degree of trismus in 60% and palatal fistula in 25% of patients; they were managed by passive stretching and dental plates, respectively. The palatal fistula problem has been further eliminated by recent modification of palatal incision; analyses on their quality of life showed satisfactory results [116, 117]. In concurrence with other reports [118], satisfactory long-term results with 5-year local salvage of 65% and EFS of 54% could be achieved [119].

Treatment of Metastatic Disease Treatment should be individualized as there is marked heterogeneity in prognosis among patients with distant metastases. Toh et al. [120] proposed a prognostic index score basing on metastasis at diagnosis, disease-free interval, performance status, and hemoglobulin level. The median OS ranged from 8 for the poor to 20 months for the good prognostic group. Hui et  al. [121] showed that patients with lung metastasis alone had a relatively favorable prognosis, a more aggressive approach should be considered. In addition to chemotherapy, surgical resection and/or high-dose RT may be considered in selected patients with limited metastases. Cisplatin-based chemotherapy is the mainstay of treatment for majority of distant failures, the most common regimen CDDP/FU [122] achieved an overall response rate (ORR) of 66% and median time to progression (TTP) of 8 months.

Fig. 28.4  Schematic computed tomography showing on (left): the recurrent tumor marked by (T) and the osteotomies by the dotted line; (right): the maxilla attached to the anterior cheek flap swung laterally to expose the recurrent tumor (T) for salvage nasopharyngectomy

394

A.W.M. Lee et al.

Addition of different drugs like bleomycin, epirubicin, ­mitomycin, methotrexate to CDDP (with or without FU) failed to achieve substantial benefits, but incurred a high incidence of hematologic toxicities and even mortality [123–126]. Table 28.7 summarizes the response and major toxicities of different regimens; cross-series comparison is impossible because of difference in patient characteristics and previous treatment. Among the modern cytotoxic drugs, studies on combination of CDDP/docetaxel [127], CDDP/capecitabine [128], and CDDP/Gemcitabine [129] reported ORR of 63–73% and median TTP 6–11 months. The latter two combinations are particularly attractive because of moderate toxicity and easy administration; docetaxel should be used with caution because of very high incidence of neutropenic toxicity [127, 130]. Using a triplet combination of gemcitabine, paclitaxel, and carboplatin for 6 cycles followed by weekly FU and folinic acid for 48 weeks, Leong et al. [131] reported an ORR of 86%, the median TTP was 8 months and the OS was up to 22 months; but hematological toxicities occurred in >79% of patients. The efficacy of molecular-targeted therapy for NPC remains uncertain. Although overexpression of epidermal growth factor receptor (EGFR) was found in more than 80% of NPC patients and this was associated with poor prog­ nosis [132], a phase II study on combination of cetuximab (a monoclonal antibody against the extracellular domain of EGFR) with carboplatin on refractory patients only achieved an ORR of 12% and TTP of 3 months [133]. Other targeted agents including Gefitinib (a tyrosine kinase inhibitor against the intracellular EGFR signaling pathway) [134] and

sorafenib (a multitargeted kinase inhibitor) [135] showed very poor response (0–4%).

Management of Late Toxicities The therapeutic margin for NPC is notoriously narrow, late toxicities following conventional 2D and 3D conformal RT have been extensively reported [136–138]. The problem of xerostomia has decreased substantially in modern IMRT era [39, 40, 71]. However, late toxicities at other sites remain a concern, especially with increasing use of concurrent CRT; swallowing problems and damage to carotid vessels become increasingly recognized. Torrential epistaxis from ruptured pseudoaneurysm at the petrous portion of the internal carotid arteries is one of the most serious toxicities [139, 140], urgent endovascular occlusion or stenting is needed for life saving. Another potentially fatal complication is stenosis of the extracranial portion of carotid arteries; Cheng et al. [141] detected severe stenosis with ³70% occlusion in 16% of long survivors, ultrasound screening was advocated for elderly patients (especially those with cardiac/cerebrovascular symptoms), carotid endarterectomy or percutaneous transluminal endoplasty may be indicated. Temporal lobe necrosis (TLN) is reported even with IMRT [140, 142, 143]. The risk increases with dose escalation [24], hypofractionation or rapid acceleration [22], and re-irradiation [100–104, 106–108]. Treatment includes anticonvulsants for temporal lobe epilepsy and steroid for gross

Table 28.7  Chemotherapy/molecular-targeted therapy for metastatic/recurrent nasopharyngeal carcinoma Author No. Cytotoxic/targeted drugs ORR % (complete) Median TTP months Toxicity % (grade ³3) Au et al. [122] Su et al. [123]

24 25

CDDP + FU CDDP + FU + Bleomycin

66 (12) 40 (4)

8 NR

Neutropenia 42 Neutropenia 36 Infections 32 Treatment death 12 Yeo et al. [161] 27 Carbo + paclitaxel 59 (11) NR Neutropenia 32 (with fever 3) Chua et al. [127] 19 CDDP + docetaxel 62 (6) 6 Neutropenia >79 (with fever 42) Ngan et al. [129] 44 CDDP + gemcitabine 73 (21) 11 Granulocytopenia 37 Wang et al. [159] 39 vinorelbine + gemcitabine 36 (3) 5 Neutropenia 44 Thrombocytopenia 18 Leong et al. [131] Carbo + gemcitabine + paclitaxel 86 (11) 8 Neutropenia 79 Anemia 32 Thrombocytopenia 29 Li et al. [128] 48 CDDP + Capecitabine 62 (6) 8 Neutropenia 15 HFS 4 Chan et al. [133] 59 Carbo + cetuximab 12 (0) 3 Overall 52 Elser et al. [135] 26 Sorafenib 4 (0) 2 Lymphopenia 17 Fatigue 7 ORR overall response rate, TTP time to progression, CDDP Cisplatin, FU Fluorouracil, Carbo carboplatin, HFS hand–foot syndrome, NR not reported

28  Management of Nasopharyngeal Carcinoma

increase in intracranial pressure; surgery is often reserved for severely symptomatic patients [144, 145]. Palsy of the last four cranial nerves (especially the twelfth) has been reported following 2D RT [136] especially among those with additional parapharyngeal boost. This could lead to swallowing difficulties and aspiration pneumonia. Damage to the pharyngo-oesophageal axis is another cause for dysphagia following IMRT [43]. The most common toxicity is hearing impairment. High tone sensorineural deafness is related to the dose to the inner ear, use of concurrent CDDP and age [138]. The mean dose to cochlea should preferably be £47 Gy especially for patients treated by CRT [146]. It should be noted that sudden deafness in long survivors might be a different problem related to vascular insufficiency and treatment with vascular expander like Dextran 40 was suggested [147]. Another problem is otitis media with effusion caused by Eustachian tube dysfunction, repeated myringotomy and aspiration can temporarily reduce the inflammation of the middle ear, prophylactic grommet insertion is not recommended as this would invite ascending infection [148]. Endocrine dysfunction remains a common toxicity following IMRT [142]. Regular endocrine assessment is indicated as hormonal replacement, mostly thyroxine or cortisol, might be required. Other complications include osteoradionecrosis [149], and radiation-induced malignancies [150, 151] with poor prognosis despite surgery.

Fig. 28.5  Exploring the possibility of further improvement by volumetric arc technique

395

Conclusion Medical progress in the battle against NPC is one of the most gratifying successes. This peculiar cancer was invariably lethal before the advent of megavoltage RT. With improving knowledge and technology, representative series from Hong Kong showed that the 5-year DSS steadily increased from 50% for patients treated from 1976 to 1985 [152] to 80% for those treated from 1996 to 2000 [153]. Together with decreasing incidence, our age-standardized mortality rate has steadily decreased from the peak of 14.1 in 1983 to 5.8 per 100,000 male populations in 2006. Continuous search for more potent systemic therapies, refinement of RT technique and precision are still demanded. Technological advances bring exciting opportunities; it is highly hopeful that these progresses can enable efficient delivery of high quality RT (Fig. 28.5). Novel therapies, for instance, recombinant adenovirus-p53 in concurrence with RT [154], open new frontiers for exploration. Furthermore, early detection and more accurate prognostication for personalized medicine are crucial for future improvement; concerted efforts in translational researches will become increasingly important.

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28  Management of Nasopharyngeal Carcinoma 122. Au E, Ang PT. A phase II trial of 5-fluorouracil and cisplatinum in recurrent or metastatic nasopharyngeal carcinoma. Ann Oncol. 1994;94:87–9. 123. Su WC, Chen TY, Kao RH, et al. Chemotherapy with cisplatin and continuous infusion of 5-fluorouracil and bleomycin for recurrent and metastatic nasopharyngeal carcinoma in Taiwan. Oncology. 1993;50:205–8. 124. Siu LL, Czaykowski PM, Tannock IF. Phase I/II study of the CAPABLE regimen for patients with poorly differentiated carcinoma of the nasopharynx. J Clin Oncol. 1998;16:2514–21. 125. Hasbini A, Mahjoubi R, Fandi A, et al. Phase II trial combining mitomycin with 5-fluorouracil, epirubicin, and cisplatin in recurrent and metastatic undifferentiated carcinoma of nasopharyngeal type. Ann Oncol. 1999;10:421–5. 126. Taamma A, Fandi A, Azli N, et al. Phase II trial of chemotherapy with 5-fluorouracil, bleomycin, epirubicin, and cisplatin for patients with locally advanced, metastatic, or recurrent undifferentiated carcinoma of the nasopharyngeal type. Cancer. 1999;86:1101–8. 127. Chua DT, Sham JS, Au GK. A phase II study of docetaxel and cisplatin as first-line chemotherapy in patients with metastatic nasopharyngeal carcinoma. Oral Oncol. 2005;41:589–95. 128. Li YH, Wang FH, Jiang WQ, et al. Phase II study of capecitabine and cisplatin combination as first-line chemotherapy in Chinese patients with metastatic nasopharyngeal carcinoma. Cancer Chemother Pharmacol. 2008;62:539–44. 129. Ngan RK, Yiu HH, Lau WH, et  al. Combination gemcitabine and cisplatin chemotherapy for metastatic or recurrent nasopharyngeal carcinoma: report of a phase II study. Ann Oncol. 2002;13:1252–8. 130. McCarthy JS, Tannock IF, Degendorfer P, et al. A Phase II trial of docetaxel and cisplatin in patients with recurrent or metastatic nasopharyngeal carcinoma. Oral Oncol. 2002;38:686–90. 131. Leong SS, Wee J, Rajan S, et al. Triplet combination of gemcitabine, paclitaxel, and carboplatin followed by maintenance 5-fluorouracil and folinic acid in patients with metastatic nasopharyngeal carcinoma. Cancer. 2008;113:1332–7. 132. Chua DT, Nicholls JM, Sham JS, et al. Prognostic value of epidermal growth factor receptor expression in patients with advanced stage nasopharyngeal carcinoma treated with induction chemotherapy and radiotherapy. Int J Radiat Oncol Biol Phys. 2004;59: 11–20. 133. Chan AT, Hsu MM, Goh BC, et al. Multicenter, phase II study of cetuximab in combination with carboplatin in patients with recurrent or metastatic nasopharyngeal carcinoma. J Clin Oncol. 2005;23:3568–76. 134. Chua DT, Wei WI, Wong MP, et al. Phase II study of gefitinib for the treatment of recurrent and metastatic nasopharyngeal carcinoma. Head Neck. 2008;30:863–7. 135. Elser C, Siu LL, Winquist E, et al. Phase II trial of sorafenib in patients with recurrent or metastatic squamous cell carcinoma of the head and neck or nasopharyngeal carcinoma. J Clin Oncol. 2007;25:3766–73. 136. Lee AW, Law SC, Ng SH, et al. Retrospective analysis of nasopharyngeal carcinoma treated during 1976–1985: late complications following megavoltage irradiation. Br J Radiol. 1992;65:918–28. 137. Lee AW, Perez CA, Law CK et al. Nasopharynx. In: Halperin EC, Perez CA, Brady LW, editors. Principles and practice of radiation oncology, 5th edn. Philadelphia: Lippincott Williams & Wilkins; 2008b. p. 820–857 138. Lee AW, Ng WT, Hung WM, et al. Major Late toxicities following conformal radiotherapy for Nasopharyngeal Carcinoma – patient and treatment related risk factors. Int J Radiat Oncol Biol Phys. 2009;73:1121–8. 139. Cheng KM, Chan CM, Cheung YL, et al. Endovascular treatment of radiation-induced petrous internal carotid artery aneurysm

399 p­ resenting with acute haemorrhage. A report of two cases. Acta Neurochir (Wien). 2001;143:351–6. 140. Kwong DL, Sham JS, Leung LH, et al. Preliminary results of radiation dose escalation for locally advanced nasopharyngeal carcinoma. Int J Radiat Oncol Biol Phys. 2006;64:374–81. 141. Cheng SW, Ting AC, Lam LK, et al. Carotid stenosis after radiotherapy for nasopharyngeal carcinoma. Arch Otolaryngol Head Neck Surg. 2000;126:517–21. 142. Kam MK, Teo PM, Chau RM, et al. Treatment of nasopharyngeal carcinoma with intensity-modulated radiotherapy: the Hong Kong experience. Int J Radiat Oncol Biol Phys. 2004;60:1440–50. 143. Bucci M, Xia P, Lee N, et al. Intensity modulated radiation therapy for carcinoma of the nasopharynx: an update of the UCSF experience. Int J Radiat Oncol Biol Phys. 2004;60:S317–318. 144. Cheng KM, Chan CM, Fu YT, et al. Brain abscess formation in radiation necrosis of the temporal lobe following radiation therapy for nasopharyngeal carcinoma. Acta Neurochir (Wien). 2000;142: 435–41. 145. Cheng KM, Chan CM, Fu YT, et  al. Acute hemorrhage in late radiation necrosis of the temporal lobe: report of five cases and review of the literature. J Neurooncol. 2001;51:143–50. 146. Young YH, Lou PJ. Post-irradiation sudden deafness. J Laryngol Otol. 1999;113:815–7. 147. Chen CY, Young YH, Hsu WC, et al. Failure of grommet insertion in post-irradiation otitis media with effusion. Ann Otol Rhinol Laryngol. 2001;110:746–8. 148. Cheng SJ, Lee JJ, Ting LL, et  al. A clinical staging system and treatment guidelines for maxillary osteoradionecrosis in irradiated nasopharyngeal carcinoma patients. Int J Radiat Oncol Biol Phys. 2006;64:90–7. 149. Dickens P, Wei WI, Sham JS. Osteosarcoma of the maxilla in Hong Kong Chinese postirradiation for nasopharyngeal carcinoma. A report of four cases. Cancer. 1990;66:1924–6. 150. King AD, Ahuja AT, Teo P, et al. Radiation induced sarcomas of the head and neck following radiotherapy for nasopharyngeal carcinoma. Clin Radiol. 2000;55:684–9. 151. Lee AW, Poon YF, Foo W, et al. Retrospective analysis of 5,037 patients with nasopharyngeal carcinoma treated during 1976– 1985: overall survival and patterns of failure. Int J Radiat Oncol Biol Phys. 1992;23:261–70. 152. Lee AW, Sze WM, Au SK, et al. Treatment results for nasopharyngeal carcinoma in the modern era: the Hong Kong experience. Int J Radiat Oncol Biol Phys. 2005;61:1107–16. 153. Pan JJ, Zhang SW, Chen CB, et al. Effect of recombinant adenovirus-p53 combined with radiotherapy on long-term prognosis of advanced nasopharyngeal carcinoma. J Clin Oncol. 2009;27:799–804. 154. Kwong DL, Pow EH, Sham JS, et al. Intensity-modulated radiotherapy for early-stage nasopharyngeal carcinoma: a prospective study on disease control and preservation of salivary function. Cancer. 2004;101:1584–93. 155. Lin S, Pan J, Han L, et al. Nasopharyngeal carcinoma treated with reduced-volume intensity-modulated radiation therapy: Report on the 3-year outcome of a prospective series. Int J Radiat Oncol Biol Phys. 2009;75:1071–78. 156. Tham IW, Hee SW, Yeo RM, et al. Treatment of nasopharyngeal carcinoma using intensity-modulated radiotherapy – The National Cancer Centre Singapore experience. Int J Radiat Oncol Biol Phys. 2009;75:1481–86. 157. Oh JL, Vokes EE, Kies MS, et  al. Induction chemotherapy followed by concomitant chemoradiotherapy in the treatment of locoregionally advanced nasopharyngeal cancer. Ann Oncol. 2003;14:564–9. 158. Wang CC, Chang JY, Liu TW, et al. Phase II study of gemcitabine plus vinorelbine in the treatment of cisplatin-resistant nasopharyngeal carcinoma. Head Neck. 2006;28:74–80.

400 159. Al-Amro A, Al-Rajhi N, Khafaga Y, et  al. Neoadjuvant chemotherapy followed by concurrent chemo-radiation therapy in locally advanced nasopharyngeal carcinoma. Int J Radiat Oncol Biol Phys. 2005;62:508–13. 1 60. J ohnson FM, Garden AS, Palmer JL, et  al. A phase I/II study of neoadjuvant chemotherapy followed by radiation

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Chapter 29

Multidisciplinary Management of Oropharynx Carcinomas Beth M. Beadle and David I. Rosenthal

Abstract  The demographics, evaluation, and treatment of patients with oropharynx cancers have changed dramatically in the last decade. Compared to other head and neck malignancies, which are declining, oropharynx cancers are increasing in numbers, younger patients are being diagnosed, and more women are developing the disease; human papilloma virus (HPV) has been implicated as a likely causative factor in these notable changes. As the nature of the disease itself changes, so does the evaluation and treatment. The integration of detailed imaging modalities, including MRI and PET scans, has helped delineate the extent of disease and guide therapy, and the molecular analysis of tumor tissue for HPV infection has helped predict the outcomes for these patients. As late as the 1990s, the standard therapy for oropharynx cancer at most institutions was surgery with postoperative radiation therapy; during this time, the majority of oropharynx carcinomas were thought to be related to tobacco use. Now, advances in radiation therapy, chemotherapy, and our understanding of a potential viral etiology of these tumors that portends a better prognosis, have caused definitive radiation therapy and chemoradiation therapy to become the standard of care. As our understanding of this disease has improved, the integration of novel targeted therapies, including the anti-epidermal growth factor receptor inhibitors such as cetuximab, has been another milestone in the treatment for these patients. Overall, our understanding of the pathogenesis and methods of treatment has undergone a significant evolution, and the integration of multidisciplinary collaboration has improved outcomes and reduced treatment toxicities for patients with oropharynx carcinomas.

Introduction

Keywords  Oropharynx cancers • Human papilloma virus • Chemoradiation • Radiation

The oropharynx is situated approximately in the middle of the upper aerodigestive tract. It is directly in communication with the other sites of the head and neck, superiorly with the nasopharynx, anteriorly with the oral cavity, and posteroinferiorly with the supraglottic larynx and hypopharynx (Fig. 29.1). Anatomically, it is bounded by the junction of the posterior extent of the hard palate superiorly, the circumvallate papillae of the tongue anteriorly, the hyoid bone inferiorly, and the pharyngeal walls in the posterolateral directions.

B.M. Beadle (*) The University of Texas M. D. Anderson Cancer Center, Radiation Oncology Department, 1515 Holcombe Blvd., Unite 97, Houston, TX 77030, USA e-mail: [email protected]

In the last decade, the evaluation and management of carcinomas of the oropharynx have undergone perhaps the most radical change of all of the head and neck malignancies. The incidence is increasing, the patient age and gender disparities are decreasing, and the prognoses are improving as human papilloma virus (HPV) infection is recognized as another major causative factor. As late as the 1990s, the standard therapy for oropharynx cancer at most institutions was surgery with postoperative radiation therapy; during this time, the majority of oropharynx carcinomas were thought to be related to tobacco use. Now, advances in radiation therapy, chemotherapy, and our understanding of a potential viral etiology of these tumors that portends a better prognosis, have caused definitive radiation therapy and chemoradiation therapy to become the de facto standard of care. As our understanding of this disease has improved, the integration of novel targeted therapies, including the anti-epidermal growth factor receptor (EGFR) inhibitors such as cetuximab, has been another milestone in the treatment for these patients. Overall, our understanding of the pathogenesis and methods of treatment has undergone a significant evolution, and the integration of multidisciplinary collaboration has improved outcomes and reduced treatment toxicities for patients with oropharynx carcinomas.

Anatomy and Lymphatic Drainage

J. Bernier (ed.), Head and Neck Cancer: Multimodality Management, DOI 10.1007/978-1-4419-9464-6_29, © Springer Science+Business Media, LLC 2011

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The oropharynx is divided into four distinct subsites for the purposes of diagnosis and treatment planning. These are: (1) the base of tongue, (2) the tonsillar complex, (3) the soft palate, and (4) the oropharyngeal walls (Fig. 29.2).

Base of Tongue The base of tongue is a muscular structure of the posterior tongue that is covered in squamous epithelium and contains numerous submucosal lymphoid nests; it is part of Waldeyer’s

Fig. 29.1  Location of the oropharynx within the head and neck and its relationship with other sites, as shown on a sagittal MRI

Fig. 29.2  Subsites of the oropharynx on (a) oral examination (b) CT scan. The subsites of the oropharynx are: a – Tonsillar complex, b – Base of tongue, c – Soft palate, and d – Oropharyngeal walls. (a) from Veasey S. Obstructive Sleep Apnea. In: Binder MD, Hirokawa N, Windhorst U eds. Encyclopedia of neuroscience. Berlin:Springer;2009. Reprinted with permission from Springer

B.M. Beadle and D.I. Rosenthal

ring. The base of tongue extends from the circumvallate papillae anteriorly to the base of the epiglottis inferiorly (including the valleculae) and to the glossopharyngeal sulci bilaterally. The base of tongue musculature is comprised of the genioglossus, styloglossus, palatoglossus, and hyoglossus muscles. The sensory innervation of the base of tongue is from the lingual branch of cranial nerve IX (glossopharyngeal), and the motor innervation is from cranial nerve XII (hypoglossal). Primary tumors of the base of tongue can grow either in an infiltrative, submucosal pattern that invade the intrinsic muscles of the tongue or in an exophytic pattern across the mucosa and into the lumen of the upper aerodigestive tract. As the tumors become larger, they may go deeply through the intrinsic muscles of the tongue and affect the extrinsic musculature, inferiorly into the structures of the hypopharynx and larynx, or laterally to the glossopharyngeal sulci and tonsils; they may also cause oral tongue immobility and deviation. Tumors of the base of tongue tend to present with advanced stages since the tongue base is largely devoid of pain fibers, and lesions are frequently asymptomatic until quite large. However, due to the innervation of the base of tongue, tumors in this region can present with referred otalgia via cranial nerve IX (glossopharyngeal) as it joins the tympanic nerve (Jacobson’s) and the two traverse the jugular foramen; this referred pain is typically deep in the ear canal. Tumors of the base of tongue frequently present with nodal metastases. The base of tongue drains to levels II, III, and IV of the neck, as well as the retropharyngeal lymph nodes. The base of tongue is a midline structure, so lymph node drainage is bilateral. Prior studies at The University of Texas M. D. Anderson Cancer Center have shown that approximately 70% of patients with base of tongue tumors will present with unilateral lymph node metastases and approximately 10–20% will present with bilateral nodal disease [1].

29  Multidisciplinary Management of Oropharynx Carcinomas

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Tonsillar Complex

Soft Palate

The tonsillar fossa is located between the palatoglossus and palatopharyngeus muscles, which when covered by their mucosa make up the anterior and posterior tonsillar pillars, respectively. The tonsils are a conglomeration of largely lymphoid tissue encased in a fibrous capsule, which rest within the tonsillar fossae. The entire region is covered in a squamous epithelium that serves as the nidus for the majority of tonsillar tumors. Tonsillar anatomy involves surface involutions, such that only a minority of the total mucosal area is visible on inspection of the surface; for this reason, a tonsillectomy is necessary for diagnosis of a potential primary tumor when no surface lesion is observed. The sensory innervation of the tonsils is branches of cranial nerve V2 (maxillary). Primary tumors of the tonsillar fossae and tonsillar pillars can either grow as exophytic lesions along the mucosal surface, spreading onto adjacent subsites, such as the soft palate, tongue base, and pharyngeal walls, or as deeply invasive lesions into the stroma in an ulcerative or endophytic pattern. Advanced tumors are capable of significant submucosal spread, including invasion into the underlying pterygoid muscles and into adjacent regions of the head and neck, including the nasopharynx, hypopharynx, and larynx. Primary tumors may arise from the tonsillar pillars, the tonsillar fossae, or the tonsils themselves; even following a tonsillectomy, tonsil tissue typically remains that may serve as the nidus for malignancy. Lesions of the tonsillar fossae and tonsils tend to present with more advanced primary disease than do those that develop in the tonsillar pillars due to the later development of overt symptoms, including pain, odynophagia, and a globus sensation. Tumors of the tonsillar region frequently present with nodal metastases. The tonsillar region primarily drains to level II of the neck, but lymph nodes in level I and the retropharyngeal nodes may also be involved. Tumors that arise from the tonsillar fossa are more likely to involve lymph nodes than those from the tonsillar pillars. In a study by Lindberg et  al., lymph node metastases were noted in 71% of patients with T1 tonsillar fossa tumors, 68% of T2 lesions, 70% of T3 lesions, and 90% of T4 lesions [1]. Another study describing patients with tonsillar tumors treated at The University of Texas M. D. Anderson Cancer Center between 1968 and 1979 demonstrated 69% of patients having lymph node metastases at presentation [2]. Since the tonsil is a lateral structure, bilateral lymphadenopathy is less common than other sites of the oropharynx. For tumors confined to the tonsillar fossa and posterior ­pillar, contralateral lymph node positivity is reported in up to 22% of cases; for tumors of the anterior pillar, this is only 6% [3].

The soft palate is a muscular structure largely covered in stratified squamous epithelium that separates the oropharynx from the nasopharynx superiorly and the oral cavity anteriorly. The soft palate musculature includes the levator veli palatine, tensor veli palatine, uvula, palatoglossus, and palatopharyngeus muscles. Anatomically, the soft palate attaches to the hard ­palate anteriorly and is contiguous with the tonsillar fossae on the lateral sides. Functionally, the soft palate is crucial in speech and swallowing. The motor innervation of the muscles of the soft palate include cranial nerve V3 (mandibular nerve) and X (vagus), which function to elevate the palate and close off the nasopharynx during swallowing and speech, preventing reflux of a food bolus superiorly and preventing breathiness and nasality of phonation, respectively termed velopharyngeal incompetence (VPI). Primary tumors of the soft palate typically arise from the squamous mucosa of the oral aspect of the soft palate. Lesions that arise from the nasopharyngeal portion are much less common. Typically, lesions grow along the mucosal surfaces and tend to be superficial. As the lesions increase in size, they may extend to the adjacent tonsillar fossae, pharyngeal walls, or the anterior palatoglossal arches. Compared to other sites of the oropharynx, lesions tend to be more symptomatic and present at earlier stages. Tumors of the soft palate predominantly drain to the lymph nodes in levels II and III, as well as the retropharyngeal nodes. In a study by Lindberg et al., clinically evident lymph nodes were present in 8% of T1 tumors, 37% of T2 tumors, 65% of T3 tumors, and 67% of T4 tumors; the overall rate of nodal metastases was 40% [1]. Given the centrality of the soft palate, bilateral lymphatic drainage is common, and bilateral nodal disease is not unusual.

Oropharyngeal Walls The oropharyngeal walls are comprised of the mucosa of the lateral and posterior aspects of the upper aerodigestive tract within the confines of the oropharynx; specifically, the posterior pharyngeal wall extends from the inferior aspect of the nasopharynx to the level of the epiglottis and the lateral pharyngeal wall extends in the same longitudinal region on the right and left aspects of the oropharynx. The oropharyngeal walls consist of a squamous mucosal epithelium that overlies the pharyngeal constrictor musculature. In turn, the pharyngeal constrictors are anterior to the retropharyngeal space, the longus capitis and colli muscles, the prevertebral fascia, and finally the vertebral bodies. The oropharyngeal walls typically are situated adjacent to the second and third cervical

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vertebrae, and this region is innervated by cranial nerves IX (glossopharyngeal) and X (vagus). Primary tumors of the oropharyngeal walls typically arise from the squamous mucosa and grow toward the lumen of the aerodigestive tract and submucosally to other sites within the oropharynx. However, it is possible for lesions of the posterior pharyngeal wall to grow into the prevertebral musculature and bony involvement of the vertebral bodies, although rare, is possible. Lesions of the lateral pharyngeal wall may also grow directly into the structures of the neck and become confluent with the lymph node basins of that region. In many cases, lesions present at an advanced stage, due to the relative paucity of early symptoms until a critical size is reached. Tumors of both the posterior and lateral pharyngeal walls primarily drain to the lymph nodes in levels II and III, as well as the retropharyngeal nodes. In a study by Lindberg et al., clinically evident lymph nodes were present in 25% of patients with T1 tumors, 30% of T2 tumors, 68% of T3 tumors, and 76% of T4 tumors [1]. An additional series, spanning 1954–1975 at The University of Texas M. D. Anderson Cancer Center, described on overall incidence of nodal disease of 57% in patients with oropharyngeal wall tumors [4]. Bilateral nodal drainage, both to the retropharyngeal and cervical nodes, is common.

Epidemiology Incidence and Mortality Tumors of the oropharynx are one of the most common types of head and neck cancer. Globally, oropharynx and hypopharynx cancer, combined, are estimated to affect over 130,000 individuals and result in over 50,000 deaths [5]. The diagnosis and outcomes of patients with these cancers, however, does significantly differ throughout the world. In men who live in developed nations, there is an estimate of 41,000 new cases per year with a mortality of 20,000, compared to an incidence of 65,000 new cases annually with a mortality of 48,000 in the developing world [5]. This discrepancy highlights the different propensities for disease development based on underlying lifestyle choices (for instance, tobacco and HPV), genetic dispositions, and preventive health measures, as well as the different standards of care throughout the world. In the United States, oral cavity and oropharynx tumors affect approximately 35,000 new patients a year and cause approximately 7,500 deaths [6]. Oropharynx tumors alone are expected to occur in approximately 5,000 patients [7]. Current statistics in the USA suggest that the incidence of

B.M. Beadle and D.I. Rosenthal

oropharyngeal cancers has continued to increase (approximately 4% per year for tonsil cancers and 2% per year for base of tongue cancers) [8]; concurrently, however, there are decreasing death rates from oral cavity and oropharynx cancers. In 1990, the estimated cancer death rate per 100,000 was 5.61; this decreased to 3.84 in 2005 [6]. This represents an absolute decrease of 1.77 per 100,000 and a percentage decrease of 31.55%. The underlying reasons for the reduced death rate are ultimately unknown, but may be due to improved screening and diagnosis, improved treatment of these malignancies, or possibly improved prognosis of virally-related tumors. In 2009, it is estimated that the patients with oropharynx and oral cavity cancers will present with largely locoregional disease. Overall, 33% of patients present with localized disease, 51% with regional disease, and only 10% with metastatic disease [6]. This highlights the opportunities for intervention for these patients, for whom locoregional disease is the common presentation and presents a unique opportunity for cure.

Changing Demographics and Risk Factors Historically, oropharyngeal carcinoma has predominantly affected older men, with 70-80% of patients being male and an average age of 50–70 years at presentation [6]. In recent years, however, the demographics of oropharyngeal cancers have changed. Multiple studies from Western Europe and the USA have suggested less gender disparity and decreased average age of presentation, with more and more patients presenting under 45 years of age [9]. These observations prompted a variety of investigations, and even though the specific trends varied from country to country, similar changes were seen worldwide in both oral cavity [10–12] and oropharynx cancers [13–15]. One of the strongest associations in carcinogenesis is the link between the development of oropharyngeal carcinomas and the use of tobacco and alcohol products. Primary studies have suggested that smoking increases the risk of head and neck squamous cell carcinomas by 12 times in women and 5 times in men [8]. Furthermore, a synergistic effect has been seen between tobacco and alcohol use [16, 17]. A pooled analyses of 17 European and American case-control studies suggested there was a greater than multiplicative effect between tobacco and alcohol use, with a population attributable risk for head and neck cancers of 35% for tobacco and alcohol combined [18]. In addition to alcohol and tobacco consumption, other lifestyle factors and sexual habits have been implicated in the pathogenesis of oropharyngeal carcinomas. The use of marijuana [19–21], dietary intake of fruits and vegetables

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[22, 23], body mass index [24, 25], and oral hygiene [26, 27] have all been studied with relation to the development of oropharynx carcinoma. Although the data have been somewhat mixed, patients who have a low dietary intake of fruits and vegetables and poor oral hygiene seem to have higher rates of oropharynx carcinomas. One of the most interesting developments in the field of head and neck cancers has been the link between oropharyngeal carcinomas and HPV infection. Although this has been well-established in the cervical cancer literature, the possible etiologic contribution of viral infection to oropharyngeal carcinomas is a more recent realization [28]. In one review of 60 individual studies, the average rate of HPV-DNA positivity was 35.6% for oropharyngeal carcinomas; this is compared to approximately 20% in other oral cavity and larynx tumors [29]. In addition, oropharyngeal cancers tend to be associated with high-risk HPV subtype 16 (87%) in contrast to cancers of the uterine cervix, which tend to be associated approximately equally with subtypes 16 and 18 [30]. Further studies have shown that patients who developed oropharyngeal cancers below 55 years of age were found to have higher risk sexual behaviors and more HPV-positive tumors than those who developed cancers at a more advanced age [30]. Oral HPV infection itself, like anogenital infection, has been found to correlate with sexual behavior, for instance the number of oral sex partners [31, 32], number of lifetime sexual partners, young age at first coitus, and a history of genital warts [33]; all these factors have been shown to correlate with an increased risk of oropharyngeal cancer in individual studies. Larger case-control studies have not demonstrated significant associations between sexual behavior and oropharynx cancers; however, these are likely rendered insignificant by the more traditional patients with oropharyngeal carcinomas, attributable to alcohol and tobacco, and a minority of patients with HPV-related tumors [34, 35]. In addition to noting an association between HPV infection and the development of oropharynx carcinomas, patients with HPV-related tumors appear to have a better prognosis than those with non-HPV-related tumors. Fakhry and colleagues reported the outcomes of patients with HPV-related oropharynx tumors compared to non-HPV-related tumors as part of an Eastern Cooperative Oncology Group (ECOG) phase II prospective clinical trial (E2399) [36]. In this population, 40% of patients had genomic DNA of oncogenic HPV in the nuclei of the tumor cells. Patients with HPV-positive tumors had improved response after induction chemotherapy (82% vs. 55%, p = 0.01) and after chemoradiation (84% vs. 57%, p = 0.007) as well as improved overall survival at 2 years (95% vs. 62%, p = 0.005). An analysis of the HPVpositivity in tumors of patients treated on RTOG H-0129, a phase III randomized trial of chemoradiation with either standard or altered fractionation, demonstrated HPV-16 positivity in 60.6% of oropharynx tumors [37]. Similar to the

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ECOG study, patients with HPV-positive oropharynx tumors had improved 2-year overall survival (87.5% vs. 67.2%). These studies have been pivotal in establishing the role of HPV-positivity in both pathogenesis and prognosis for patients with oropharynx cancer. The scientific link between oropharyngeal cancers and HPV is now well-recognized, and the data suggesting improved responsiveness to both chemotherapy and radiation in HPV-positive tumors are compelling. Indeed, future clinical trials addressing treatment for these malignancies are being designed separately for patients with HPV-positive and HPV-negative tumors. The philosophical approach being developed is to intensify treatment for those patients with HPV-negative tumors, and therefore poorer prognoses, and to evaluate patterns of failure and minimize overall toxicity for those with HPV-positive tumors, and therefore better prognoses.

Pathology and Pathogenesis The large majority of tumors that arise in the oropharynx are squamous cell carcinomas; less than 10% of tumors are of a different histology, with minor salivary gland adenocarcinomas, lymphomas, melanomas, sarcomas, and undifferentiated cancers making up this group [38, 39]. In addition, benign conditions, including papillomas, fibromas, hemangiomas, neuromas, and cysts, may occur in the oropharynx and must be considered in the differential diagnosis. Finally, metastases to the oropharynx, although rare, have been described [40, 41]. Given the preponderance of squamous cell carcinomas, the remainder of this chapter concentrates on their diagnosis and management.

Role of Pathological Assessment Tissue diagnosis and adequate pathologic assessment are crucial to the diagnosis of head and neck squamous carcinomas. Tissue can be obtained through core needle biopsies, excisional, or incisional biopsies. Conventional hematoxylin and eosin staining remains key to the diagnostic evaluation. A variety of features can be described in squamous carcinomas diagnosed through this analysis; however, the relative importance of other features of the tumor has been the subject of significant debate. Perineural invasion [42, 43], basaloid features [44], and keratinization [45] have all been investigated as potentially important prognostic features. More recently, studies suggest that basaloid features and nonkeratinizing tumors may reflect HPV status and confer their improved prognosis [46].

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In addition to traditional histologic appearance, the tissue from oropharynx squamous cell carcinomas also can be analyzed for characteristic molecular markers. In situ hybridization analysis for high-risk HPV subtypes is increasingly used as part of the pathologic assessment of squamous cell carcinomas of the oropharynx (Fig. 29.3a); this, however, is a technique that requires specific training and expertise. Immunohistochemical analysis (IHC) for p16 overexpression has also proven useful; studies begun in HPV-positive cervical cancer specimens demonstrated a correlation bet­ ween HPV-infection and p16 overexpression [47]. Now, viral infection with HPV is correlated with expression of p16 in carcinomas of the head and neck. Some studies suggest that p16 overexpression is indicative of better prognosis, similar to HPV-positivity [48]; in contrast, p16 is downregulated in tobacco-related cancers. IHC analysis for p16 often demonstrates diffuse positive staining in HPV-positive tumors (Fig.  29.3b); this is a relatively inexpensive and efficient test. Since HPV-associated tumors have been shown to have  a better prognosis than non-HPV-associated tumors, this testing may have a significant impact on treatment decisions.

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Pathogenesis of Oropharyngeal Carcinoma Squamous cell carcinomas of the oropharynx are considered the result of multiple events at the molecular level; each of these events may reflect a change due to a genetic predisposition or an exposure to an exogenous environmental agent [49]. Multiple independent events that cause the loss or inactivation of tumor suppressor genes and activation of oncogenes appear crucial to the development of oropharyngeal carcinomas; environmental agents can cause specific damage or trigger cascades that contribute to these pathways. Elegant studies of genetic alterations in squamous cell tumor specimens from the head and neck by Califano and colleagues have suggested a model of genetic progression in these lesions (Fig. 29.4). The most common alteration is the loss of chromosomal region 9p21, a region that encodes two suppressors p16 and p14ARF; this abnormality is present in over 70% of dysplastic lesions, suggesting that its loss is an early event in the carcinogenic pathway [49–51]. Another early genetic abnormality is loss of a region of chromosome 3p, which encodes two suppressor genes FHIT and RASSFIA [51–53]. Later genetic events appear to include loss of

Fig. 29.3  Specialized pathologic assessment of oropharynx tumors. (a) HPV in situ hybridization demonstrating nuclear staining in a tonsil squamous cell carcinoma (b) p16 immunohistochemical analysis showing diffuse staining of a base of tongue squamous cell carcinoma

Fig. 29.4  Schematic of genetic alterations contributing to the development of squamous cell carcinoma

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heterozygosity (LOH) of chromosome region 17p and p53 mutation [54]. In aggregate, these genetic events contribute to genomic instability and the development of aneuploidy; this progression has been shown to be crucial in the progression of normal mucosa to dysplasia and finally to invasive carcinoma. Exogenous environmental factors appear to contribute to this cascade in a variety of ways. Carcinogen exposure, such as use of tobacco and alcohol, can cause direct genetic insult or act indirectly through mucosal damage. Damage of the mucosa may trigger inflammatory cascades that involve COX-2 and EGFR activation, Cyclin D1 activation, and increased proliferation; this compensatory mechanism to the acute injury increases proliferation and puts the mucosa at increased risk of mutation [55]. Viral infection with high-risk HPV subtypes exerts direct influence on the pathways of carcinogenesis in oropharyngeal carcinomas. Most HPV-related cancers carry the viral DNA integrated into the cellular chromosomes at one or more loci [56, 57]. It is believed that expression of two early genes in the viral genome, E6 and E7, are crucial to viral mediated cancer development. The E6 protein, mediated by a cellular protein called E6-associated protein (E6AP), forms a complex with the tumor suppressor p53, causing degradation of p53 via ubiquitin-mediated proteolysis [58]. The ability to inhibit the tumor suppressor activity of p53 has been shown to reduce the ability of the cell to respond to genotoxic stress [59] and genetic instability [60]. The E7 protein interferes with the activity of the protein product of the retinoblastoma gene, which is a tumor suppressor that is involved in cellcycle control and progression; in this way, the E7 protein can disrupt signals that would normally stop DNA synthesis and cell-cycle progression [61–63]. The molecular effects of both E6 and E7 in HPV-associated cells are believed to contribute to the transformation of infected cells to carcinoma.

Multidisciplinary Initial Assessment and Staging The Role of History and Physical Examination The primary evaluation of a patient with oropharyngeal cancer is a comprehensive history and physical examination. On history, the patient’s symptoms depend highly on the location and extent of the tumor. Patients with early stage oropharyngeal carcinomas may present with few symptoms; the tumors may have been found incidentally on scans for other indications or dental evaluations. One common presenting symptom of oropharynx cancer is a painless neck mass, representing lymph node metastasis;

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in many cases, only after a full examination is a primary identified. When patients do develop symptoms due to local disease, pain is often the earliest to develop. This may be pain at the site of the primary or referred pain to the middle ear. The latter occurs via the pharyngeal and tonsillar branches of cranial nerve IX, which traverse the petrosal ganglion and then synapse with the tympanic nerve of Jacobson, which innervate the middle ear. As tumors progress, odynophagia, dysphagia, dysarthria, and trismus may develop and cause the patient to seek medical attention. The physical examination is a crucial part of the evaluation for oropharyngeal cancer patients, and it highly affects treatment decisions and planning. The head and neck examination should evaluate the local extent of the primary tumor and the presence and location of lymph nodes. Inspection of the oropharynx can be performed by direct or indirect laryngoscopy or fiberoptic nasopharyngolaryngoscopy; there should be a complete evaluation of all mucosal surfaces to ensure there are no other lesions and to fully appreciate the extent of the primary tumor. In addition, digital examination is crucial to the estimate of the primary disease size. Often, there can be infiltrative processes that are underappreciated by both inspection of the mucosal surface and segmental imaging. A full evaluation of the adjacent oral cavity should be performed to understand whether the tumor invades these areas. Attempts should be made to estimate the size of the primary lesion, its limits of spread, and associated lymphadenopathy, since all of these contribute to the ultimate staging and treatment recommendations. Depending on the subsite of the primary tumor, 45–78% of patients may present with cervical adenopathy at the time of diagnosis [1]; assessment of the lymph nodes is crucial to an accurate understanding of the extent of disease. While level II is the most common lymph node station affected, the other cervical nodal areas, as well as the supraclavicular fossae, should be assessed. Finally, associated symptoms, such as tongue deviation, tongue fixation, trismus, and sensory impairment, should be evaluated; these suggest further extension of the tumor that will influence stage and treatment recommendations. Cranial nerves V, VII, XI, X, and XII are especially at risk for compromise due to invasion by oropharyngeal cancers, and these should be specifically assessed during the physical examination. In the case of an inadequate physical examination, the patient may require an examination under anesthesia (coupled with biopsy) to fully appreciate the extent of disease and establish a diagnosis. Pretreatment dental evaluation is also crucial to the ultimate management of patients with oropharyngeal carcinoma. Treatment of oropharyngeal cancer with radiation has several short- and long-term effects on dentition and oral health, and an evaluation of the baseline dentition is crucial to effective management. Patients who develop xerostomia are at greater risk for dental caries and demineralization. The decrement in

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perfusion following radiation therapy leads to greater difficulty in healing from dental procedures. A clinical dental evaluation and radiographic studies should be done to assess the status of the teeth. Prior to radiation therapy, any non-restorable teeth should be extracted. In addition, the patient will benefit from lifetime dental fluoride prophylaxis. Long-term dental care by a dentist familiar with the effects of radiation therapy should be undertaken following treatment [64]. Finally, patients with oropharyngeal cancer should be assessed for the status of their general health, in a comprehensive manner, by the team of treating physicians; in many cases, an evaluation by an internist may be beneficial. Many of these patients have medical comorbidities, such as diabetes or hypertension, and many are at risk for secondary malignancies. In addition, patients are at risk for chemical hypothyroidism after treatment with radiation therapy to the neck; baseline thyroid function should be evaluated prior to treatment and then monitored appropriately in follow-up. A chest radiograph, complete blood count, and serum chemistry evaluation, in addition to a review of their past history, will provide a better understanding of their disease and baseline health status. Finally, lifestyle interventions, such as smoking cessation, are crucial for long-term success.

The Role of Imaging Advanced imaging techniques are standard in the evaluation and staging of oropharyngeal tumors. The goal of imaging is to establish the extent and size of the primary tumor, evaluate nodal disease, and identify perineural spread and bony destruction. The optimal type of imaging for head and neck cancers depends on the site of disease and goals of the evaluation. Computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasound

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(US) are all complementary modalities and can be used to evaluate different aspects of the disease (Fig. 29.5). Standard imaging evaluation for an oropharynx tumor includes CT or MR imaging of the head and neck, with intravenous contrast (unless medically contraindicated), to evaluate the primary tumor and nodal disease. CT is considered by many to be preferable to MRI for the imaging of oropharynx tumors because it is less affected by breathing and swallowing artifacts [65], although some centers prefer MRI due to their expertise with this modality. The imaging of oropharyngeal tumors on CT or MRI is quite variable, and enhancement may or may not reflect the full extent of disease. It is important to correlate the mass and enhancement observed on imaging with the clinical examination in order to fully appreciate the extent of disease. Bone invasion and destruction is well-delineated on CT scan, and hence CT may indicate more extensive disease than previously appreciated. Despite the benefits of CT, it is limited by artifacts caused by metallic dental implants and fillings; angled cuts may be helpful in providing more useful images through important areas. If these maneuvers are not sufficient, MRI may be used, since it is not affected by metallic artifact. Imaging of nodal disease can be accomplished with CT, MRI, PET, or US. Clinical evaluation and cross-sectional imaging is estimated to be negative in 15–25% of patients with true nodal metastases from head and neck cancers [66]. However, patients with head and neck cancers often can have reactive adenopathy that does not reflect metastatic disease; hence, false positives and false negatives are clinically relevant. With modern techniques, CT and MRI are considered equivalent in the detection of nodal disease from head and neck cancers [67]; CT is typically the primary modality used